Table of Content

30 August 2014, Volume 32 Issue 4

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From Recognition to Practice：The 140th Anniversary of the Discovery of Clonorchis sinensis

QIAN Men-Bao, CHEN Ying-Dan, ZHOU Xiao-Nong-*

2014, 32(4):  1-247-252. 

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It has been the 140th anniversary since the discovery of Clonorchis sinensis， of which adult worms were found by McConnell in an oversea Chinese in Calcutta， India, September of 1874. Then， Japanese scholar Kobayashi proved that freshwater fish served as the second intermediate hosts in 1910， while another Japanese parasitologist Muto found that the first intermediate hosts were freshwater snails in 1918. However， the perniciousness has not been recognized until recently. C. sinensis infection was classified as definite carcinogen（group 1） in cholangiocarcinoma in 2009 by the International Agency for Research on Cancer， WHO， and listed as one of the 17 diseases in WHO’s first report on neglected tropical diseases in 2010， while its disease burden was published on line in an international journal in 2011. Nevertheless， our awareness on and practices in the control of clonorchiasis still lag behind the reality. Great efforts on research and control of clonorchiasis are especially required in China， since China takes the biggest share in global disease burden of clonorchiasis.

Construction of Plasmodium falciparum Signal Peptide Peptidase-GFP Mutant and its Expression Analysis in the Malaria Parasite

LI Xue-rong1，2 *，WU Yin-juan1，2，SHANG Mei1，2，LI Ye1，2，XU Jin1，2，YU Xin-bing1，2，ATHAR Chishti3

2014, 32(4):  2-253-257. 

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Objective  To construct recombinant plasmid pSPPcGT which contains signal peptide peptidase gene of Plasmodium falciparum （PfSPP） and GFP， and transfect into P. falciparum（3D7 strain） to obtain mutant parasites which can express PfSPP-GFP. 　Methods  Plasmodium falciparum（3D7 strain） genomic DNA was extracted from cultured malaria parasites. The C-terminal region of PfSPP， an 883 bp gene fragment was amplified by PCR， and then cloned into pPM2GT vector to get recombinant vector pSPPcGT. The recombinant vectors were identified by PCR， double restriction enzyme digestion and DNA sequencing. pSPPcGT vector was transfected into malaria parasites. The positive clones were selected by adding inhibitor of Plasmodium falciparum dihydrofolate reductase WR99210 to the culture medium. The pSPP-GFP-transfected parasites were fixed with methanol， stained with DAPI， and observed under immunofluorescence microscope. The PfSPP-GFP expression in P. falciparum was identified by SDS-PAGE and Western blotting.  Results   The C-terminal region of PfSPP was amplified from P. falciparum（3D7 strain） genomic DNA by PCR with the length of 883 bp. The constructed recombinant vectors were identified by PCR screening， double restriction enzyme digestion and DNA sequencing. The pSPPcGT vector was transfected into P. falciparum and the positive clones were selected by WR99210. GFP fluorescence was observed in transfected parasites by immunofluorescence microscopy， and mainly located in the cytoplasm. The PfSPP-GFP expression in malaria parasites was confirmed by Western blotting with a relative molecular mass of Mr 64 000.  Conclusion  Recombinant vector PfSPP-GFP is constructed and transfected into P. falciparum to obtain P. falciparum mutant clone which can express PfSPP-GFP.

Construction of HSP47-shRNA and its Impact on HSP47 at Cellular Level

FAN Xiang-Xue-1, DAO Ran-1, HUANG Jia-Quan-1*, DENG Ju-Gong-2, LI Lan-1, MA Ke-1, XU Lei-1, NING Qin-1

2014, 32(4):  3-258-263. 

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Objective  To construct a short hairpin RNA（shRNA） against HSP47 gene， assess the expression level of HSP47 gene in NIH/3T3 cells， and observe the influence on cell function.  Methods  The HSP47-shRNA sequence presented at the downstream of the U6 promoter. The shRNA expression constructs were created using PCR-based methods. The PCR product was digested with NheⅠ/HindⅢ and ligated into pGCsi/U6/Neo vector to produce HSP47-pGCsi-U6-shRNA（HSP47-1-pGCsi-U6-shRNA，HSP47-2-pGCsi-U6-shRNA and HSP47-3-pGCsi-U6-shRNA）. The non-interference vector and non-related interference vector served as control. The vectors were transfected into NIH/3T3 fibroblast cells by liposome mediated gene transfection method. Transfection efficiency and fluorescence intensity were determined by fluorescence microscopy at 12， 24， 48， and 72 hours after transfection， respectively. Cells were collected before transfection, and at 24， and 48 hours post-transfection， respectively， HSP47 mRNA and protein expression levels were assessed by real-time PCR and Western blotting. The mRNA expression of TGF-β1， collagen types Ⅰ and Ⅲ in NIH/3T3 cells， and TGF-β1 levels in cell culture supernatant were determined.  Results  HSP47-shRNA vector was transfected into NIH/3T3 cells by liposome-mediated transfection. The transfection efficiency in each HSP47-shRNA plasmid interference group was about 60.0%， and there is no statistical difference among the interference groups（P>0.05）. A small amount of green fluorescent cells were found at 12 h post-transfection. The number of green fluorescent cells increased with the transfection time， and reached strongest at 72 h after transfection. shRNA interference significantly inhabited HSP47 expression in NIH/3T3 cells. At 24 h after transfection with HSP47-1-shRNA， the inhibition effect was the strongest， and the relative silence efficiency of HSP47 mRNA was（25.83±1.79）%， lower than that of control group and non-related group（P＜0.05）. Collagen synthesis and secretion by NIH/3T3 cells reduced significantly at 24 and 48 hours post-transfection with HSP47-1-shRNA； and there was a significant difference between HSP47-1-shRNA intervention group and non-related controls. After transfection for 24 and 48 h， mRNA expression of collagen types Ⅰ and Ⅲ decreased to （56.52±1.64）% and （53.48±2.54）%， （54.17±2.63）% and （50.21±2.34）%， respectively， significantly lower than that of the control group and non-related group （P<0.05）； however， no significant difference was found among the interference groups（P>0.05）. In each HSP47-shRNA plasmid interference group， TGF-β1 mRNA expression was the lowest at 24 h post-transfection. The relative mRNA expression level was（63.23±2.18）%， （64.5±3.17）%， and（75.19±4.20）% in HSP47-1-shRNA， HSP47-2-shRNA， HSP47-3-shRNA groups（P>0.05）， respectively， lower than that of the control group and non-related group（P<0.01）. At 24 and 48 h post-transfection， TGF-β1 expression was the lowest at 24 h post-transfection， and the relative expression level in HSP47-1-shRNA， HSP47-2-shRNA， HSP47-3-shRNA groups was（51.79±3.12）%， （66.67±2.13）%， and （69.61±3.65）%， respectively. Compared with control group， the expression of TGF-β1 in HSP47-1-shRNA and HSP47-2-shRNA2 groups was significant inhibited， but there was no significantly difference between control group and HSP47-3-shRNA group（P＞0.05）.  Conclusion  HSP47-shRNA interference plasmid is constructed. HSP47-shRNA effectively inhibits protein expression of HSP47， and results in changes of cell function.

Construction of β-hydroxyacyl-acyl Carrier Protein Dehydratase Mutant of Toxoplasma gondii by Tetracycline Inducible Expression System

TUN Liang, XUE Lan-Lan, WANG Xiao, TUN La-Mei, JIANG Xu-Gan, CHEN Cheng-Xia-*

2014, 32(4):  4-264-267. 

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  Objective To construct a β-hydroxyacyl-acyl carrier protein dehydratase（FABZ） mutant of Toxoplasma gondii with tetracycline inducible expression system. Methods The fabz gene was amplified from T. gondii genomic DNA, and then used to construct the tetracycline inducible expression vector pTetO7-Sag1-FABZ-Ty-DHFR. The vector was transfected into TATi strain by electroporation. The FABZ defective mutant was selected by pyrimethamine and limiting dilution assay. The expression of Ty-tagged mutant was detected by Western blotting. 5×105 tachyzoites of FABZ defective mutant were cultured in HFF in the presence of anhydrotetracycline （ATc, 1 μg/ml） for 24 h and 48 h, respectively. The expression of Ty-tagged FABZ protein in the mutant was detected by Western blotting. Results The mutant could express the transit peptide （t-FABZ） and mature FABZ （m-FABZ） with the Ty-epitope tag. After ATc added in culture medium for 24 h and 48 h, the expression of t-FABZ in the mutant decreased significantly（P<0.05）. Conclusion The FABZ mutant is constructed with a tetracycline inducible expression system.

Experimental Study on the Der f 1 mRNA Molecules Derived from Dermatophagoides farinae for Specific Immunotherapy on Murine Model of Asthma

JIANG Yu-Xin-1 *, YIN Kang-2, JIN Wen-Jie-2, TUN Lou-Yi-2, LI Chao-Pin-3

2014, 32(4):  5-268-273. 

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Objective To investigate the effect of Der f 1 mRNA molecules for specific immunotherapy on murine model of asthma. Methods Fifty BALB/c mice were randomly divided into 5 groups： PBS group， Der f 1 sensitization group， Der f 1 specific immunotherapy（SIT） group， β-actin mRNA SIT group， and Der f 1 mRNA SIT group. On days 0， 7 and 14， mice in PBS group received PBS injection； mice in the other groups were intraperitoneally injected with 10 μg Der f 1. At day 21， the mice in the 4 experimental groups were challenged with a 30-min inhaled dose of Der f 1（100 μg/ml） for 7 successive days. Two weeks after the final sensitization， the mice in the above five groups were immunized by intradermal injection with PBS， 1 μg Der f 1， 10 μg Der f 1， 2 μg β-actin mRNA， and 2 μg Der f 1 mRNA， respectively for 3 times at one-week intervals. Two weeks after the last intradermal injection， all mice were sacrificed and bronchoalveolar lavage fluid （BALF） was collected. ELISA was performed to detect the levels of IFN-γ and IL-13 in BALF， the number of eosinophils in the BALF was recorded. Splenocytes were prepared， and cultured with Der f 1 allergen （10 μg/ml） for 72 h. Splenocytes of PBS group was cultured without Der f 1 allergen. The levels of IFN-γ and IL-13 in splenocyte culture supernatant were measured by ELISA， as well as serum antibody levels of total IgE， allergen-specific IgE （sIgE）， sIgG1， and sIgG2a. Lung sections were stained in hematoxylin and eosin, and observed under the microsope. Results Except for PBS group， mice in the other 4 group showed symptoms of acute asthma attack. Compared with Der f 1 sensitization group ［（897.56±105.73） pg/ml］ and β-actin mRNA SIT group ［（219.47±64.72） pg/ml］， the level of IFN-γ in BALF from Der f 1 mRNA SIT group ［（897.56±105.73） pg/ml］ and Der f 1 SIT group ［（864.48±70.62）pg/ml］ significantly increased（P<0.01）. However， the level of IL-13 in BALF from Der f 1 mRNA SIT group ［（241.64±31.41） pg/ml］ and Der f 1 SIT group ［（321.94±41.07）pg/ml］ was significantly lower than that of Der f 1 sensitization group ［（520.62±43.77） pg/ml］ and β-actin mRNA SIT group ［（507.22±42.26） pg/ml］（P<0.01）. The number of eosinophils in Der f 1 mRNA SIT group ［（1.33±0.44）×105/ml］ and Der f 1 SIT group ［（1.48±0.39）×105/ml］ was also lower than that of Der f 1 sensitization group ［（3.54±0.52）×105/ml］ and β-actin mRNA SIT group ［（2.98±0.53）×105/ml］（P<0.01）. The levels of IFN-γ and IL-13 in splenocyte culture supernatant showed that IFN-γ level in Der f 1 mRNA SIT group ［（420.91±69.92） pg/ml］ and Der f 1 SIT group ［（334.92±43.72） pg/ml］ was significantly higher than that of Der f 1 sensitization group［（123.75±15.48） pg/ml］ and β-actin mRNA SIT group［（128.84±59.00） pg/ml］ （P<0.01）. However， IL-13 level of Der f 1 mRNA SIT group ［（268.51±40.42） pg/ml］ and Der f 1 SIT group ［（285.26±62.21）pg/ml］ was significantly lower than that of Der f 1 sensitization group ［（613.89±51.54） pg/ml］ and β-actin mRNA SIT group ［（524.05±39.12） pg/ml］（P<0.01）. Compared with Der f 1 sensitization group ［total IgE： （94.34±11.66） ng/ml， sIgE： （65.67±9.47） ng/ml， sIgG1： （75.18±9.52） ng/ml， sIgG2a： （2.81±1.17） ng/ml］ and β-actin mRNA SIT group［total IgE： （86.48±10.26） ng/ml， sIgE： （62.36±8.35） ng/ml， sIgG1： （69.51±8.98） ng/ml， IgG2a： （1.06±0.11） ng/ml］， the serum antibody levels of total IgE ［（33.72±9.78） ng/ml］， sIgE ［（22.76±8.09） ng/ml］， sIgG1 ［（17.87±7.59） ng/ml］ of Der f 1 mRNA SIT group decreased significantly（P<0.01）， whereas the level of IgG2a ［（7.74±0.88） ng/ml］ increased（P<0.01）. Compared with Der f 1 sensitization group， the asthmatic symptoms were relieved after immunization with Der f 1 mRNA for specific immunotherapy， including intact structure of respiratory and alveolar epithelial cells， decreased inflammatory cell infiltration， and similar to those in Der f 1 SIT group. However， the breakage and detachment of bronchial epithelial cells occurred in β-actin mRNA SIT group. Conclusion Der f 1 mRNA vaccine can correct Th1 and Th2 imbalance.

Construction of Suppression Subtractive Hybridization cDNA Library of Half-blood Males of Dermacentor silvarum and Analysis of Differentially Expressed Genes

LIU Qi-1, WANG Wei-Lin-2, MENG Qiang-Feng-2, XU Zhan-1, CUI Ji-1, LIU Xin-Xin-1, WANG Wei-Li-2 *

2014, 32(4):  6-274-279. 

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  Objective To construct a suppression subtractive hybridization（SSH） cDNA library of half-blood males of Dermacentor silvarum， and analyze the differentially expressed genes. Methods Total RNA was extracted from the half-blood males and unfed males of D.　silvarum. cDNA was synthesized following the protocol of SMARTER cDNA synthesis kit. After RsaⅠdigestion， cDNA was ligated to adaptors. The cDNA from the half-blood males was used as the tester， and unfed males as the driver. The SSH library was constructed using TaKaRa PCR-select cDNA subtraction kit. Differentially expressed cDNAs were amplified by nested PCR， cloned into PMD-18T vector， transformed into E. coli DH5α, and the white-blue plaque selection was used to get the positive clones. The titer of SSH library and the recombination efficiency were calculated. Individual colonies were randomly selected from library. Subtractive efficiency of the subtracted cDNA library was examined by reverse Northern blotting and RT-PCR. Positive clones with differentially expressed genes were sequenced. Homology comparison and function prediction were performed by Blastn and Blastx. Results The bands of double-stranded cDNAs from half-blood males and unfed males of D. silvarum were dispersed and longer than 500 bp. After RsaⅠ digestion， the ds cDNA-fragments were 100-1 000 bp. The ligation reaction efficiency of adaptor was more than 25%. Nested PCR showed that the bands of subtracted ds cDNA were gathered， ranging from 250 to 500 bp. The titer of SSH library was 700 000 pfu/ml， and the recombination efficiency was 88.5% （239/270）. Reverse Northern hybridization revealed that the clones showed stronger signals in half-blood males cDNA probes than in unfed males cDNA probes. RT-PCR showed that among the eight random selected positive clones， 5 clones were up-expressed under half-blood condition. A total of 87 differentially expressed sequence tags（ESTs， 200-800 bp） were obtained from 115 positive clones. Among the 87 ESTs， 53 ESTs showed sequence similarities to genes from other tick species， and 34 were homologous with genes from other insects. The main biological function of obtained ESTs were related to blood sucking and digestion， such as energy metabolism， signal transduction， and transcription regulation. Conclusion The SSH cDNA library of half-blood male Dermacentor silvarum is constructed. The differential expressed genes are related to blood sucking and digestion.

Effect of Immunotherapy with Recombinant Allergen Group 3 from Dermatophagoides farinae in Asthma Mice

LI Na-1, JIANG Yu-Xin-2, DIAO Ji-Dong-1, DIAO Bei-Bei-1, LI Chao-Pin-1*

2014, 32(4):  7-280-284. 

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Objective  To explore the effect of specific immunotherapy with major 3 group recombinant allergen rDer f 3 of Dermatophagoides farinae in murine asthma model.  Methods  Forty BALB/c mice were randomly divided into 4 groups， namely PBS group（negative control）， ovalbumin（OVA） group（positive control）， rDer f 3 allergen sensitization group（asthma group）， and rDer f 3 specific immunotherapy group（SIT group）. The mice in asthma group and SIT group were injected intraperitoneally with purified rDer f 3 protein on days 0， 7 and 14， respectively， and rDer f 3 solution was inhalated from day 21 for 7 days. During the 25th-27th day， mice in SIT group were injected subcutaneously with 100 μg rDer f 3 allergen for 30 min before nasal inhalation. Mice in groups of PBS and OVA were treated with PBS and OVA， respectively. Twenty-four hours after the final challenge， all mice were sacrificed， the bronchoalveolar lavage fluid（BALF） was collected and the total number of white blood cells and the number of eosinophils were recorded. The levels of IL-5 and IFN-γ in BALF and supernatant of cultured splenocytes were detected by ELISA， as well as the serum levels of specific IgE and IgG2a antibodies. Lung tissues were stained with haematoxylin and eosin for histological analysis.  Results  Compared with the asthma group， the rDer f 3-induced lung inflammation was significantly alleviated in SIT group. The total number of white blood cells［（7.03±1.38）×10⁸/ml］ in SIT group was considerably lower than that of OVA group［（22.11±3.70）×10⁸/ml］ and asthma group［（22.75±3.24）×10⁸/ml］（P＜0.01）. The change trend of eosinophil leukocytes was similar with that of white blood cells. IL-5 levels in BALF［（108.20±11.02） pg/ml］ and splenocyte culture supernatant ［（98.34±13.06） pg/ml］ in SIT group were significantly lower than that of OVA group［（182.04±13.94） pg/ml，（208.26±10.63） pg/ml］ and asthma group［（195.33±15.33） pg/ml，（179.54±13.65） pg/ml］（P＜0.01）. Whereas， the level of IFN-γ in BALF［（107.98±12.64） pg/ml］ and supernatant of cultured splenocytes［（105.51±11.62） pg/ml］ in SIT group was significantly higher than those of OVA group and asthma group（P＜0.01）. Compared with OVA group[（26.87±4.30）IU/ml] and asthma group[（35.25±8.84）IU/ml]， a lower level of allergen-specific IgE［（9.12±3.78）IU/ml］ and higher level of allergen-specific IgG2a［（38.52±6.33） μg/ml］ were observed in SIT group（P＜0.01）.  Conclusion  rDer f 3 allergen can reverse allergen-induced airway and lung inflammation in murine asthma model.

Experimental Infection of Galba pervia，Radix swinhoei and Physa acuta with Fasciola hepatica in Dali，Yunnan

FANG Wen-*, LI Tian-Mei, LI Ke-Rong, CHEN Feng, LIU Yu-Hua

2014, 32(4):  8-285-288. 

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Objective  To determine the intermediate host of Fasciola hepatica in Dali of Yunnan Province， and investigate its development and characteristics.  Methods  F. hepatica eggs from cattle were collected from July 2012 to July 2013， and placed in 28 ℃ water bath for incubation. Galba pervia， Radix swinhoei， and Physa acuta were collected from Dali， and used to be infected with F. hepatica in the laboratory. Trematode infections were excluded from the snails before experiment. All the snails were infected with F. hepatica miracidia， reared in mud pots. Dead snails were dissected for observing the development of F. hepatica. The metacercariae were collected and identified by PCR amplification of partial sequence of mitochondrial cytochrome oxidase subunit I（COX1） gene.  Results  A total of 1 146 R. swinhoei， 996 P. acuta， and 3 307 G. pervia snails were infected with F. hepatica， respectively. Mother rediae were found in two R. swinhoei snails， but no child rediae were observed in the snails. No larval forms were found in P. acuta. G. pervia was infected by F. hepatica with an infection rate of 27.2%（900/3 307）. The miracidium escaped from the egg and penetrated into G. pervia at temperature 22 ℃， developed into a sporocyst after 7-15 days， which transformed into mother redia at the 11st-20th day post?鄄infection. The mother redia developed into daughter redia at the 30th-37th day， and produced cercaria with longtail， and became metacercaria at the 42nd-55th day. PCR confirmed that the metacercariae were that of F. hepatica， with an obvious band（approximately 500 bp）.  Conclusion  Among the three potential intermediate hosts in Dali， G. pervia is experimentally infected with F. hepatica.

Immunoreactivity and Immunogenicity Analysis of  the Recombinant Cathepsin L-like Protease of Fasciola hepatica in SD Rats

DAN Xu-Hua, WEN Xiao-Bei-*, WANG Chun-Ren, LI Xiao-Juan, WEI Xiao-Man

2014, 32(4):  9-289-292. 

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Objective  To analyze the immunoreactivity of recombinant cathepsin L-like proteases（CatL） protein of Fasciola hepatica and its immunogenicity in SD rats.  Methods  The E. coli BL21（DE3） cells harbouring recombinant plasmid pET30a-FhCatL were inoculated in LB medium， and the protein expression was induced with IPTG. The recombinant protein FhCatL was analyzed by SDS-PAGE and the immunoreactivity was identified by Western blotting with sera from Fasciola hepatica-infected goat as the primary antibody. Twenty SD rats were randomly divided into immunized group and adjuvant control group. SD rats in immunized group were injected subcutaneously with 200 μg of purified FhCatL protein. All the rats received three immunizations at 3-week intervals. The adjuvant control group with 10 SD rats received only adjuvants emulsified with the same amount of PBS. Serum samples were collected at the day before the second and final immunization， 3， 6， and 9 weeks after the final immunization. The IgG antibody of rats′ sera was examined by indirect ELISA and spleen lymphocyte proliferation（SLP） was tested by methyl thiazolyl tetrazolium（MTT）. Results The molecular weight of purified FhCatL was about Mr 42 000. The recombinant FhCatL was recognized by pool sera of goats naturally infected with F. hepatica. The titer of specific antibody IgG in SD rats induced by the recombinant protein against CatL protein was significantly higher than that of the control， and the antibody titer reached the peak at three weeks after the final immunization（1 ∶ 102 400）. The stimulation index of splenocytes in immunized group was 2.176±0.047， which was significantly higher than that of the control（1.171±0.032）（P<0.05）. Conclusion The recombinant FhCatL protein bears stronger immnoreactivity and immunogenicity.

Morphological Characteristics and Gene Expression of Chrysomya megacephala Eggs in Different Developmental Stages

JIA Bing-1, LIU Yu-Ming-2, WANG Qi-Yan-1, ZHANG Gong-Ling-1, WANG Jie-1, DAI Jia-Lin-1, HUANG Jiang-1*

2014, 32(4):  10-295-298. 

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 Objective  To research the morphological characteristics and differential gene expression of Chrysomya megacephala eggs in different developmental stages.  Methods  After C. megacephala laid eggs（0 h）， the eggs were collected every 2 h until eggs hatched into larvae. The morphological characteristics of C. megacephala eggs in different developmental stages were observed by stereo microscopy and scanning electron microscopy. The total RNA of the fly eggs was extracted. The expression levels of bicoid， slalom and chitin synthase genes was determined by Real-time flourescence quantitative PCR. Statistic analyses were performed with SPSS 19.0.  Results  Under the stereomicroscope， at 0-4 h after egg laying， the morphological change of C. megacephala eggs was not obvious. At the 6th hour after egg laying， somites were formed. After 8 hours the eggs shriveled. At the 9th hour after egg laying， the eggs hatched into larvae. The scanning electron microscope images showed that the morphological change of eggs was not obvious in the first 4 hours， the end of micropyle slightly outward， the surface around the micropyle was smooth. At the 6th hour after egg laying， the end of micropyle began to sag and irregular protrusions formed around the micropyle. At the 8th hour the end of micropyle was obviously dented. After 9 hours larvae hatched from eggs. Real-time fluorescence quantitative PCR indicated that the expression levels of bicoid， slalom and chitin synthase genes from C. megacephala eggs regularly changed with the developmental stages. There was a significant difference in threshold cycle values among the three genes（P＜0.05）. Conclusion  The morphological characteristics of C. megacephala eggs change with the development stage. The levels of gene expression in different development period of C. megacephala eggs are different.

Effect of the Excretory/Scretory Proteins from Trichinella spiralis on Apoptosis of NCI-H446 Small-cell Lung Cancer Cells

CHANG Gong-Min-1, DIAO Lei-1, WANG Xiao-Jie-2, FANG Yan-Hui-1, LI Dan-1, LUO Jing-Mei-1, DU Lian-Yang-1 *

2014, 32(4):  11-299-303. 

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Objective To investigate the effect of excretory/secretory proteins from Trichinella spiralis on apoptosis of NCI-H446 small-cell lung cancer cells. Methods Trichinella spiralis muscle stage larvae （5×106/ml）were cultured in culture media for 24 h， the excretory/secretory proteins were collected from the supernatant of culture media. NCI-H446 small-cell lung cancer cells（No. A05） were randomly divided into three groups： experiment group（A）， standard control group （apoptosis group， B）， and control group （C）. NCI-H446 cells in groups A and B were cultured with 0.3 mg/ml T. spiralis excretory/secretory proteins， and 6.4 μg/ml cisplatin for 24 h， respectively. NCI-H446 cells of group C were cultured for 24 h without any treatment. The expression of Bcl-2， Fas and Fasl mRNA was detected by RT-PCR. C-myc protein expression level was examined by Western blotting and immunofluorescence assay. Results The level of Bcl-2 mRNA was lowest in group A（0.575±0.047） , Bcl-2 mRNA level in group C （0.975±0.069） was higher than that of group B （0.850±0.073）（P<0.05）. Fas mRNA level was highest in group A（0.975±0.115）， followed by group B（0.817±0.121） and group C（0.769±0.061）（P<0.05）. The level of Fasl mRNA in groups A， B， and C was 0.669±0.051， 0.787±0.124， and 0.875±0.125， respectively（P<0.05）. Fas/Fasl mRNA ratio in groups A， B， and C was 1.475，1.038，and 0.878. Western blotting showed that the expression of C-myc protein in group C（1.172±0.026） was highest， followed by group B （1.074±0.069） and A（0.566±0.054）（P<0.05）. Immunofluorescence test indicated that the C-myc protein was found in the cytoplasm and the nucleus 24 h after treated with 0.3 mg/ml T. spiralis excretory/secretory proteins and 6.4 μg/ml cisplatin. Conclusion Trichinella spiralis excretory/secretory proteins may inhibit apoptosis of NCI-H446 small-cell lung cancer cells by reducing the apoptosis protein C-myc and Bcl-2 mRNA levels， and causing the increase of Fas/Fasl mRNA ratio.

Functional Change of Dendritic Cells/Regulatory T Cells/T-helper 17 Cells and the Mechanism of its Cross-regulation in Malaria

CHEN Guang-*, LIU Lei

2014, 32(4):  12-304-307. 

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The cross-regulation of dendritic cells （DC）/regulatory T cells （Treg）/T-helper cells （Th17） is a complex process that involves multiple signals. During Plasmodium infection， the interaction of DC， Treg， and Th17 influence the cross modification， and induce activation of DC/Treg/Th17 to regulate Th1/Th2 polarization and control the dynamic balance of Th immune response by cytokines. This article summarizes the change in cell function of DC/Treg/Th17 and the mechanism of its cross-regulation in malaria.

Advances on Antitumor Effect of Parasites

WANG Su-Wen, SUN Jun-*

2014, 32(4):  13-308-310、315. 

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Immune response induced by parasites could inhibit tumor growth and promote apoptosis of tumor cells. The investigation into this character will provide new insights on the anti-tumor effect of parasites. The mechanism of parasite immune evasion may provide a reference for tumor research. Furthermore， some anti-parasitic drugs have shown antitumor effect indicating that the development of antitumor drugs may get inspiration from anti-parasitic drug studies.

Role of Macrophages in Schistosome Infection

FANG Yan, CHEN Qing, TUN Chen-Yun, WANG Zhao-Jun-*

2014, 32(4):  14-311-315. 

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Each stage of schistosome in human body can cause disease. Pathogenic factors released by the parasites induce host immune responses and cause a series of immunopathological changes. As a major cell population in innate immunity, macrophages are important in the initiation and development of schistosomiasis. This paper summarizes the activation and polarization of macrophages, and the role of macrophages in schistosome immunopathology and immune evasion.

Prevalence of Metacercariae of Clonorchis sinensis in Wild Freshwater Fishes from Nenjiang River around Qiqihaer City

LIU Ji-xin1 *, SUN Yan-hong1, ZHANG Hao1, LI Chao-pin2

2014, 32(4):  15-292-294. 

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From May to November 2013, a total of 1 175 wild freshwater fishes were collected from the rivers of Chuoer, Yalu, Wuyuer, Alun, and Yin in Nenjiang River basin Qiqihaer City, and examined for metacercariae by direct compression method. The metacercariae were collected by artificial digestion method. Forty Kunming mice were infected with 30-40 metacercariae of Clonorchis sinensis. The mice were sacrificed 36 days after infection, and the adult worms were collected from bile duct, and observed under microscope. The results showed that a total of 1 175 fishes, belonging to nine species were taken from the Nenjiang basin of Qiqihaer region. The infection rate of Clonorchis sinensis metacercariae was 51.2% （602/1 175）. All the species were infected besides Silurus asotus, and the highest prevalence （82.7%, 91/149） was found in Longnose gudgeon and the lowest（7.1%, 6/84） in Perccottus glenii. Among the rivers, the highest prevalence of metacercariae was in Wuyuer River（65.7%, 218/332）, and the lowest was in Alun River and Yin River（24.1%, 67/278）（P＜0.05）. Each part of the body in the Carassius auratus and Pseudorasbora parva were susceptible for metacercariae. The main infection site in Longnose gudgeon was the fish scales, and C. sinensis metacercaria was first discovered in the brain tissue of Phoxinus lagowskii. The experimental results showed that the adult worms of C. sinensis were found in the hepatic bile duct of the mice, with an infection rate of 85.0% （34/40）. The suckers, digestive system and reproductive system of C. sinensis were visible clearly.

Diagnosis of an Imported Falciparum Malaria Case by Fluorescence Quantitative PCR

ZENG Yong，CAI Ai-hong，ZHU Hai-bo，NI Xiao-mei，KE Zhen

2014, 32(4):  16-316-317. 

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The whole blood sample from a patient with imported falciparum malaria was tested by microscopic examination and fluorescence quantitative PCR. Microscopic examination results showed that Plasmodium falciparum parasites were found in the thick and thin blood films with a low parasite density of 240 parasite/μl of blood. The specific DNA fragment of P. falciparum was amplified by fluorescence quantitative PCR， and this case was further confirmed as P. falciparum infection.

An Analysis on Pf60.1 Gene Polymorphism of Plasmodium falciparum from Imported Cases

WU Kai，ZHOU Shui-mao，YANG Yan，WANG Chong-xin，XU Ming-xing

2014, 32(4):  17-318-319. 

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One hundred and one imported falciparum malaria cases in Wuhan City were confirmed by microscopy and Nest-PCR， and the blood samples were collected. The Pf60.1 gene was amplified by PCR. Among 101 blood samples， three Pf60.1 fragments ［313 bp （56.5%， 52/92）， 340 bp （37.0%， 34/92）， 313 bp+340 bp （6.5%， 6/92）］ were amplified from 92 samples. Among 83 blood samples from patients returning from Africa， 313 bp fragment were found in 46 samples （55.4%， 46/83）， 340 bp fragment were found in 31 samples （37.1%， 31/83）， and 7.2% （6/83） was mixed-fragment （313 bp+340 bp）. Among 9 samples from southeast Asia， 6 samples were with 313 bp fragment and 3 samples with 340 bp fragment. The results indicated that the most common genotype was 313 bp-genotype， and there would be polyclonal P. falciparum infections.

Epidemiological Analysis of Imported Malaria in Xiaogan City from 2008 to 2013

LYU Bin1，LI Hao2 *，MOU Hong-jie1，HUANG Jing1

2014, 32(4):  18-320-321. 

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The epidemiological features of 27 imported malaria cases during 2008-2013 in Xiaogan were analyzed. Among the cases， 21（77.8%） were falciparum malaria，and 6（22.2%） were vivax malaria. Twenty-five（97.3%） cases were among 19-47 year-old males. The reported cases increased from 2010 to 2013， and there was no significant difference among seasons. The cases were reported from all the seven counties. Eleven cases（40.7%） were reported by Xiaonan District. Twenty-three cases were infected in Africa.

Preparation and Identification of Polyclonal Antibody against Small Peptides of β-tubulin of Toxoplasma gondii

MAO Xiao-Yong-1, HUA Qian-Qian-2, LI Xiang-Zhi-2, TAO Li-Li-2, TAN Feng-2 *

2014, 32(4):  19-322-323. 

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The amino acid sequences of β-tubulin from Toxoplasma gondii stains （GT1 and ME49） and human were aligned by ClustalW2 software. Based on the alignment result， the C-terminal peptides of β-tubulin of T. gondii were artificially synthesized. Rabbits were immunized with 0.5 mg synthesized peptides for five times at 2-week intervals. Serum samples were collected at the second week after the final immunization， and were analyzed for specific antibodies by ELISA. Finally， the specific-β-tubulin polyclonal antibody was evaluated by Western blotting with the total protein of RH strain， ME49 strain， and PRU strain of T. gondii， respectively. The results showed that β-tubulin of T. gondii stains（GT1 and ME49） shared 100% amino-acid sequence identity， and there was 98% amino acid homology between T. gondii and human. The main variable region was the C-terminus. After the fifth immunization， the titers of polyclonal antibody reached 1 ∶ 52 800. Western blotting result indicated that the specific-β-tubulin polyclonal antibody reacted with β-tubulin in all the three strains （RH， ME49， and PRU）， respectively.

Dynamic Changes of Serum Antibody Titer in a Rabbit Model of Acanthamoeba Keratitis

WANG Ru-Hua-1, ZHENG Wen-Yu-2, DIAO Li-Wei-1, FENG Xian-Min-1 *, LI Yao-1, JU Xiao-Gong-1

2014, 32(4):  20-324-326. 

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Ten healthy New Zealand white rabbits were randomly divided into two groups named as experiment group（n=8） and normal control group（n=2）. Left eyes were for experiment, right eyes served as control. New Zealand rabbits were each injected by subconjunctival route with hydrocortisone for three days, and then Acanthamoeba keratitis was induced by intrastromal injection of live Acanthamoeba healyi trophozoites and cysts. Eyes in control group were injected with equivalent volume of physiological saline. Corneal lesions of rabbits were recorded every day after injection, etiological diagnosis was carried out by corneal scraping. Blood samples were examined for serum antibody titer by ELISA. Corneas were removed for pathological examination. Corneal scraping and corneal histopathologic examination proved that experiment eyes were infected by Acanthamoeba, and appeared typical manifestations and pathological changes of Acanthamoeba keratitis. Serum antibody titer raised continuously with infection time and reached the highest level（A450 value=2.2358） on the 28th days post-infection, then began to decline and remained higher level than the control until the rabbits were sacrificed. In control group, no significant change in antibody titer had taken place.