Objective To observe the destroyed architecture of splenic lymphoid follicles in mice infected with Schistosoma japonicum by immunohistochemistry. Methods The mice infected with S. japonicum(20 cercariae/mouse)for 8 weeks were sacrificed,and the splenic samples were paraffin embedded and sliced. The sections were first stained by hematoxylin and eosin to observe the massive structure of splenic lymphoid follicles,and then B cells,follicular dendritic cells(FDC)and germinal center cells were labeled with anti-B220,anti-CD21 or anti-Ki67 antibodies respectively by immunohistochemistry to observe the distribution of the specific cells of lymphoid follicles. Results The results of HE staining showed that the structure of lym-phoid follicles in spleens of infected mice was blurred,the number and area of follicles were significantly reduced compared to those of the normal mice. The immunohistochemical staining showed that the splenic T/B lymphocyte segregation ,FDC network and germinal centers of the infected mice all disappeared. Conclusion The structure of splenic lymphoid follicles in the mice infected with S. japonicum is obviously damaged.

ObjectiveTo discuss the clinical value of serum CA125 and endometrial antibody (EMAb) for the diagnosis of endometriosis.Methods216 patients were determined by the presences of CA125 and EMAb before operation.ResultsAll cases were diagnosed by pathology after operation. CA125 positive rate in the endometriosis group was 58.3% and that in the control group was 12.5%. The difference between two groups was significant (P<0.01).EMAb positive rate in the endometriosis group was 31.3% and that in the control group was 14.3%. The difference between two groups was also significant (P<0.01). When determining CA125 alone to diagnose endometriosis, the sensitivity rate was 58.3% and specificity rate was 87.5%. If determining EMAb alone to diagnose endometriosis, the sensitivity rate was 31.3% and specificity rate was 85.7%. When one of them was used as diagnostic criterion, the sensitivity and specificity were 64.6% and 73.2% respectively. If combining use of both CA125 and EMAb as diagnostic criterion, the sensitivity and specificity were 25.0% and 100% respectively.Conclusions The determination of serum CA125 or EMAb levels is helpful for the qualitative diagnosis of endometriosis, especially using them combined, the diagnostic accuracy may be enhanced.

Objective:To establish Crx-iCreERT2 fluorescent reporter human embryonic stem cell lines using CRISPR/Cas9 technology and 3D retinal organoid culture.Methods:The target site sequence of H9 cell line was verified by polymerase chain reaction (PCR). SgRNAs were designed by CRISPR/Cas9 technique and their activity was detected.The most optimal sgRNA was selected according to the factors such as activity and specificity.After identification of the target vectors by restriction enzyme and sequencing, the target vectors were transferred to the H9 cell line by electroporation.P2A-tdTomato-P2A-iCreERT2 was inserted between Exon4 and 3’-untranslated region of hES-ZLM-001 gene.Knockin positive clones were obtained after drug treatment, enrichment of positive clones.Primers were designed to perform PCR on the target region, and homozygous de-resistant knockin positive cell clones were selected according to the sequencing results and peaks.The 1-A07 cell line was cultured, and then flow cytometry for the proportion of OCT4 positive cells, immunofluorescence for three stem cell molecular markers including SOX2, NANOG, SSEA4, karyotype analysis were carried out to confirm whether the 1-A07 cell line could be used for further experiments.Retinal organoids were obtained by three-dimensional (3D) culture technology and the expression of molecular markers was detected by immunofluorescence at different developmental stages of retinal organoids. Results:The target site sequence of H9 cell line was consistent with that given by Genebank and Ensembl.Sixteen sgRNAs were designed according to the target site sequence of H9 cell line, and finally sgRNA8 and sgRNA12 were selected.The sgRNAs and recombinant plasmids were transfected into the H9 cell line by electroporation, and four homozygous de-resistant knockin positive cell clones were obtained by PCR.Crx-iCreERT2 fluorescent reporter human embryonic stem cell lines were successfully obtained.In 1-A07 cell line, the proportion of OCT4 positive cells was about 98.7% by flow cytometry, and the expression of three stem cell markers was positive by immunofluorescence, and the karyotype was normal 46, XX.The results showed that the 1-A07 cell line could be used for further experiments.The Crx-iCreERT2 fluorescent reporter human embryonic stem cell lines were differentiated into tdTomato positive retinal organoids by 3D culture technology.BRN3A positive ganglion cells, CALBINDIN positive horizontal cells and CHAT positive amacrine cells appeared on day 30 of differentiation.RECOVERIN positive photoreceptors arose on day 45 of differentiation.PKCα positive bipolar cells presented on day 90 of differentiation.Ganglion cells were shown in the deep layer of retinal organoids, and horizontal cells, amacrine cells and bipolar cells in the middle layer, and photoreceptors in the top layer.Conclusions:Crx-iCreERT2 fluorescent reporter human embryonic stem cell lines are successfully established and can be differentiated into retinal organoids that express tdTomato red fluorescence through 3D culture technology.Those retinal organoids contain the same types of neurons as normal human retinas, and follow a certain temporal and spatial developmental sequence similar to the developmental rules of normal human retinas.Crx-iCreERT2 fluorescent reporter human embryonic stem cell line is a powerful tool for researching retinal development and diseases and can be applied in treatments for blindness.

Background and purpose:We investigated whether lfuorine-18 lfuorodeoxyglucose (18F-FDG) maximal standard uptake value (SUVmax) of the primary tumor (SUV-T), SUVmax of the regional lymph nodes (SUV-N) or the overall loco-regional lesion SUVmax (SUV-TOTAL) was related to survival of patients with stage Ⅲ non-small cell lung cancer (NSCLC) who received Cetuximab and combined definitive chemoradiotherpay. Methods:From September 2009 to July 2012, seventeen patients with unresectable stageⅢNSCLC receiving cetuximab with cisplatin/vinorelbine (NP) followed by concomitant NP and intensity-modulated radiotherapy (IMRT) at the Fudan University Shanghai Cancer Center were enrolled onto a prospectively study. All patients received positron emission tomography/computerized tomography (PET/CT) scans within 2 weeks before enrolment. Univariate analysis were used to assess the correlation between SUV-T, SUV-N, SUV-TOTAL, gender, age, histology, tumour-node-metastasis (TNM) stage, performance status (PS) as well as smoking status and survival. The factors which showed statistical signiifcance entered into multivariate Cox-regression model. Survival functions of different populations were estimated by Kaplan-Meier method and compared by Log-rank test. Results:In the univariate analysis, SUV-T, SUV-N, SUV-TOTAL, PS and smoking status were prognostic factors. The best cut-off values for SUV-T, SUV-N and SUV-TOTAL were 11, 11 and 20, respectively. Multivariate analysis revealed that SUV-TOTAL (P=0.012), SUV-T (P=0.025), and SUV-N (P=0.033) were independent predictors of survival with hazard ratio (HR) of 14.7, 11.2, and 6.2, respectively. Conclusion:Local, regional and locoregional maximal SUVs deifned by 18F-FDG PET-CT scanning may have a strong correlation with survival in this patients setting, which merits further study.

Objective To evaluate the effectiveness of thoracic electrical bioimpedance(TEB)in monitoring the cardiac function of peritoneal dialysis patients.Methods One hundred and one patients with continuous ambulatory peritoneal dialysis (CAPD) and 30 healthy persons (control group)were included in the study.Thoracic electrical bioimpedance (TEB) noninvasive hcmodynamic monitoring and echocardiography were taken to analyze the correlation between indexes.Results Echocardiography showed that left atrial diameter (LAD),left ventricular end diastolic diameter (LVDd),left ventricular end systolic diameter (LVDs),interventricular septal thickness （IVST),interventricular septal thickness (PAP),left ventricle weight index (LVMI) of CAPD group were higher than that of the control group (all P ＜ 0.05),early and late wave of mitral valve flow (E/A) of CAPD group was lower than that of control group (P ＜ 0.05).TEB monitoring showed that cardiac output (CO),stroke volume (SV),acceleration index (ACI),ejection fraction (EF),velocity index (Ⅵ) of CAPD group were significantly lower than that of control group (all P ＜ 0.01),systolic time ratio (STR),SVR,TFC of CAPD group were significantly higher than that of control group (P ＜ 0.01).Correlation analysis show that left ventricular ejection fraction (LVEF) was negatively correlated with BNP (r =-0.467,P ＜ 0.01),LVMI was positively correlated with BNP (r=0.416,P ＜ 0.01),PEP,STR and TFC were positively correlated with BNP (r =0.404,P ＜ 0.01; r =0.572,P ＜ 0.01; r=0.471,P ＜ 0.01),EF was negatively correlated with BNP (r =-0.664,P ＜ 0.01).Correlation analysis between echocardiogaphy and TEB monitoring index showed there was significant correlation between EF and LVEF (r =0.451,P ＜ 0.01),SVR and TFC were positively correlated with LVMI (r =0.232,P ＜ 0.05; r =0.284,P ＜ 0.05),SV was positively correlated with E/A (r =0.285,P ＜ 0.05),pre-ejection period (PEP) and STR were negatively correlated with LVEF (r =-0.389,P ＜ 0.01; r =-0.446,P ＜ 0.01),TFC was positively correlated with LAD (r=0.279,P ＜ 0.05).Conclusion TEB monitoring can accurately evaluate the cardiac function with the advantage of dynamic monitoring and simple operation.It can partly replace the echocardiography test.

Background and purpose:Clinical data show that Endostar, a recombinant human endostatin, has the therapeutic beneift for patients with non-small cell lung cancer (NSCLC) while combined with chemotherapy or ra-diotherapy. However, the microenvironment changes induced by Endostar monotherapy in NSCLC is not yet clear. The purpose of this study was to prospectively study tumor vascular effects of Endostar monotherapy in patients with locally advanced or advanced NSCLC by dynamic contrast-enhanced perfusion computed tomography (CT perfusion, CT-p). Methods:Previously untreated patients with histologically or cytologically conifrmed locally advanced or advanced NSCLC were eligible. All patients received daily Endostar (7.5 mg?m2) for 14 days. CT-p scans were acquired at the baseline and post-treatment. CT-p parameters, such as blood lfow (BF), blood volume (BV) and permeability surface PS (area product), were measured in all patients.Results:Of all 7 patients enrolled, four were staged asⅢB and three as stageⅣ (2 with malignant pleural effusion, 1 with brain metastasis). The median BF, BV and PS values of baseline and post-treatment were 27.1/48.9 mL/100 mL/min, 86.8/84.8 mL/100 mL and 45.0/54.0 mL/100 mL/min, respectively. After administration of Endostar for 14 days , BF showed a signiifcant increase compared with that at baseline (P=0.028), whereas no signiifcant changes were found in BV (P=0.398) and PS (P=0.237) values.Conclusion:Our results suggest that Endostar monotherapy induces a signiifcant increase in BF whereas no signiifcant difference in BV and PS.

Objective To perform the cloning of the gene encoding Schistosoma japonicum Chinese-strain hypoxanthine-guanine phosphoribosyltransferase(HGPRT)and its expression in Escherichia coli.MethodsA couple of primers were designed with the BamHI restriction endonuclease site introduced in forward primer and SalI in reverse primer.Total RNA was isolated from adult worms of S.japonicum Chinese-strain(Anhui-strain,Sjc-A)and the SjcHGPRT gene was amplified by reverse transcriptase-polymerase chain reaction(RT-PCR).The PCR product and the prokaryotic expression vector pET28a were digested by both restriction endonucleases BamHI and SalI.The target DNA fragments were purified and cloned properly into pET28a.After identification by en-donucleases digestion,PCR and sequencing,the recombinant plasmid pET28a-SjcHG PRT was transformed into competent E.coli BL21 and expressed in the presence of IPTG.Results pET28a-SjHGPRT was sequenced and shown to be 99% and 83% identical in deduced amino acid sequence to that of S.japonicum Chinese-strain(Hunan-strain,Sjc-H)and S.mansoni HGPRT,respectively.The results of SDS-PAGE and Western blot revealed that the molecular weight of expressed protein was around 30 kDa and could be recognized by anti-His-G-HPR antibody and sera from mice and human with schistosomasis japonica.Conclusion The recombinant plasmid containing SjcHGPRT cDNA is successfully constructed and its expression protein(reSjcHGPRT)is also successfully purified.

Objective To clone and express the gene encoding Schistosoma japonicum myophilin-like protein (SjcMLP) and to study the antigenicity of the recombinant protein. Methods The SjcMLP gene was amplified by PCR. The PCR product was cloned into T vector, and then subcloned into expression vector pQE30. The recombinant plasmid of pQE30-SjcMLP was transformed into E.coli M15, and induced with IPTG for expression. The bacterial lysis was conducted by ultrasonication and the supernatant was analysed by SDS-PAGE. The recombinant protein (reSjcMLP)was purified with the Ni-NTA resin, and analysed with SDS-PAGE and Western blot. The titers of sera from C57BL/6 mice immunized subcutaneously with reSjcMLP were detected by ELISA. Results The results of SDS-PAGE and Western blot showed that the molecular weight of expressed fusion protein was around 24.8 kDa and was recognized by the sera from the mice infected with Schistosoma japonicum. The purified protein of reSjcMLP was coated for ELISA test and the IgG titers in the sera from the mice immunized with reSjcMLP were as high as 1∶12 800 reacted with. However, no significant difference was found in worm reduction rates between the immunized mice and control mice. Conclusions The fused recombinant protein of reSjcMLP is successfully ex-pressed and purified. The recombinant protein in this experiment fails to induce significant protection against the challenge infection in C57BL/6 mice.

Objective To clone and express the partial encoding sequence of Mr 70 000 heat shock protein of Cryptosporidium andersoni (CaHSP70) in Escherichia coli and identify the recombinant protein. Methods Total RNA was extracted from oocysts of C.andersoni isolated from Xuzhou, Jiangsu (XZ-BOV). The CaHSP70 gene was amplified by RT-PCR. The PCR product was cloned and then subcloned into pET28a vector, and the recombinant plasmids were transformed into E.coli BL21(DE3) subsequently. The expressed protein induced by IPTG was purified and identified by SDS-PAGE and Western blotting, and was further analyzed by relevant bioinformatics softwares. The specific IgG antibodies in mice immunized by rCaHSP70 were detected by Western blotting and ELISA respectively. Results The deduced amino acid sequence showed to be identical with that of C. andersoni Mr 70 000 heat shock protein (HSP70). The recombinant protein expressed in the form of inclusion body was about Mr 43 000. It could be recognized by anti-His G labeled HRP antibodies and all the sera from mice infected with C. andersoni and children infected with C. parvum as well as sera from mice immunized with rCaHSP70 respectively. The rCaHSP70 possibly had multiple domains and potential antigenic determinants. Phylogenetic analysis showed that XZ-BOV and C. andersoni were in the same clade. ELISA showed that the level of specific antibodies against rCaHSP70 in immunized BALB/c and C57BL/6 mice was significantly higher than that of mice before immunization. Conclusion The recombinant plasmid pET28a-CaHSP70 has been constructed. The purified rCaHSP70 exhibits high antigenicity and seems a potential candidate antigen for immunodiagnosis of cryptosporidiosis.

Objective To study the structural features and characteristics of a novel gene Schistosoma japonicum 79(Sj79), and observe its effect of RNA interference(RNAi),so as to provide the experimental basis for its further function study and mechanism study of anti reproductive development of schistosome. Methods The gene structure and characteristics of Sj79 were analyzed by bioinformatics methods. Then the expressions of Sj79 messenger RNA(mRNA)during the different develop?mental stages of schistosome were analyzed and the effects of RNAi silencing were observed by the soaking method. The tran?scriptional levels of Sj79 after RNAi were detected by real time PCR. Results The open reading frame of Sj79 contained 696 base pairs with an exon structure. The gene had obvious stage specificity,and its transcriptional level in mature female worms was the highest. After soaking for 3 d,the Sj79 mRNA level[(41.0 ± 12.3)%]in the siRNA?1 group with low dosage(20 nmol/L) was lower than that in the siRNA?NC group[(103.2 ± 14.4)%],the difference was statistically significant(t=3.28,P<0.05). When with high dosage(200 nmol/L ),both the Sj79 mRNA levels in the siRNA?1 group[(15.8 ± 10.9)%]and siRNA?2 group [(11.1 ± 8.8)%]were significantly lower than that in the siRNA?NC group[(100.1 ± 6.3)%](t=13.44,27.84,both P<0.01). After soaking for 7 d,only the Sj79 mRNA levels in the siRNA?1group[(43.4 ± 4.5)%]and siRNA?2 group[(62.5 ± 5.4)%]with low dosage were lower than that in the siRNA?NC group[(100.4 ± 5.2)%],and the differences had statistical sig?nificance(t=8.33,5.07,both P<0.01). Conclusion Through this study,we have improved the mRNA sequence and genom?ic information of Sj79 gene,and understood its structural features,as well as selected out two effect fragments siRNA?1and siR? NA?2 which will provide the basic evidences for the further study on egg laying interference of the female adult worm of schisto?some in vitro.