Application of circulating cell-free nucleic acid in clinical diagnosis and treatment of colorectal cancer / 国际肿瘤学杂志

The incidence of colorectal cancer (CRC) is increasing year by year, and early diagnosis is of great significance to improve the prognosis of patients. Circulating cell-free nucleic acid (cfNA) has the advantages of non-invasive, real-time monitoring, and overcoming tumor heterogeneity. The characteristics of cfNA content, mutation, methylation and fragmentation patterns provid important reference value for early diagnosis, curative effect monitoring, prognosis judgment and medication guidance of CRC. However, the practical application of cfNA in the clinical diagnosis and treatment of CRC still needs to solve the problems of unstandardized detection technology, high detection cost, screening of markers with high diagnostic efficacy, and construction of multi-combination models. These challenges will provide new directions for future cfNA research.

Application of tumor biomarkers in the diagnosis and prognosis of hepatocellular carcinoma / 国际肿瘤学杂志

Tumor biomarkers have multiple characteristics, including noninvasive, repeatable analysis and real-time monitoring, and they have important application value in early diagnosis and prognosis monitoring of hepatocellular carcinoma (HCC). In recent years, the researches on tumor markers of HCC have developed rapidly. There are not only traditional serological tumor markers, such as alpha fetoprotein, des-gamma carboxy prothrombin, Golgi protein 73, glypican-3, etc., but also new emerging "liquid biopsy" tumor markers, such as circulating tumor cells, circulating tumor DNA etc. Further study on the correlation between tumor biomarkers and HCC can provide reference for the treatment and prognosis evaluation of HCC.

Research advances on clinical applications of cell-free DNA from peripheral blood in liver cancer / 中华肝胆外科杂志

Liver cancer is a malignant tumor with the characteristics of high morbidity and mortality.However,the routine detection methods for the liver cancer such as imaging tests and pathological detection are with low specificity and sensitivity.And it's also hard to detect the condition of patients dynamically.Cell-free DNA (cfDNA) is the DNA fragment that is away from the extracellular and exists in the blood,which contains genetic information from the organism.The related disease information could be reflected on the cfDNA in patients with liver cancer,indicating that cfDNA has great potential in the screening,diagnosis,treatment and prognosis of liver cancer.The clinical applications of cfDNA can make up for the deficiency of existing detection methods and achieve noninvasive and dynamic monitoring of liver cancer.This article will present a review on the progress of clinical applications of cfDNA in liver cancer.

Association study between a genetic polymorphism in cytochrome P450 19 gene rs10046 and colon and rectal cancer / 中国医师杂志

Objective To investigate the relationship between the polymorphism of cytochrome P450 19 (CYP19) gene rs10046 and the risk of colon and rectal cancer.Methods Using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to analyze gene polymorphism in CYP 19 gene rs10046 in 198 cases of colon and rectal cancer patients (case group) and 309 cases of healthy controls (control group).The genotype frequency and relative risk of CYP19 gene rs10046 between the two groups were compared and the relationship with the clinicopathological features of colorectal cancer was analyzed.Results In case group,the prevalence rates of CYP19 rs10046 genotypes C/C,C/T and T/T were 28.3％,44.4％ and 27.3％,respectively,and 17.2％,51.8％ and 31.1％ in the control group,respectively,with statistical significant difference (P ＜ 0.05).Compared with wild-type C/C,the susceptibility of colorectal cancer with the genotypes of C/T and T/T was decreased by 0.521 (95％ CI:0.330-0.822)and 0.532 (95 ％ CI:0.322-0.880) respectively.Moveover,in the non-smoking group,the risk of colorectal cancer with genotype T/T or C/T was decreased by 0.409 (95％ CI:0.210-0.798) compared with genotype C/C.The interaction was not exist in smoking group.Conclusions The polymorphism of CYP19 gene rs10046 is related to the susceptibility of colon and rectal cancer.The T/T,C/T genotype of CYP19 rs10046 decrease the risk of colon and rectal cancer,and which might be the protective factor of colon and rectal cancer.

The current situation and development tendency of medical functional virtual experiments teaching / 中华医学教育探索杂志

Virtual experiments teaching has been characterized by openness, interactivity and re-source sharing. It efficiently improves the experiments teaching effects and promotes the teaching reform. At present the virtual experiment systems used by domestic universities can realize simulation of the ex-perimental principle, apparatus, object, operation and data. In the virtual experiment system students deepen the understanding of the experiments through foregrounding and the networked virtual experiment manage-ment effectively improves the effects of experiments teaching through behind-the-scenes action.

Clinical significance of MRI and transrectal ultrasound combined with carcinoembryonic antigen examination on accurate preoperative staging of rectal carcinoma / 肿瘤研究与临床

Objective To study the consistency in the diagnosis of preoperative TNM rectal cancer staging using high resolution magnetic resonance imaging (MRI) and transrectal ultrasound (TRUS) combined with carcinoembryonic antigen (CEA) and postoperative pathological TNM.Methods 156 cases pathologically proven were retrospectively analyzed and divided into 4 groups including preoperative MRI group (39 cases),TRUS group (39 cases),MRI and TRUS group (39 cases),MRI and TRUS combined with CEA group (39 cases).The differences between preoperative T,N staging and postoperative pathologic T,N staging were analyzed.Results There were statistically significant differences in the diagnosis of preoperative T and postoperative pathological T in 4 groups (T: Kappa =0.685,P =0.000; N: Kappa =0.544,P =0.000),but there were no significant differences in preoperative N and postoperative pathological N staging in preoperative MRI group,TRUS group,MRI and TRUS group (Kappa =0.142,P =0.329; Kappa =0.154,P =0.645; Kappa =0.154,P=0.229),and significant difference was observed in MRI and TRUS combined with CEA group (Kappa =0.544,P =0.000).There were no significant differences in the accuracy of T staging among the 4 groups (x2 =0.326,P =0.574; x2 =0.562,P =0.719; x2 =0.287,P =0.986),but significant difference in the accuracy of N staging were showed among the 4 groups (x2 =4.643,P =0.026; x2 =6.643,P =0.026; x2 =5.243,P =0.019).Conclusion Preoperative evaluation by the MRI add TRUS combined with CEA can improve the accuracy of preoperative staging,which can provide more reliable basis for decision-making and improve the coincidence rate of operative procedures in line with the estimate.It also provides the basis fur the accurate preoperative diagnosis and individualized treatment.

Construction and secretory expression in E.coli of anti-HAb18G molecule ScFv gene / 中国免疫学杂志

Objective:To construct a gene of ScFv against human HAb18G molecule and secretively express it in E coli Methods:The V H and V L genes cloned from McAb HAb18 hybridoma cell were ligated with a flexible linker(Gly4Ser) 3 to construct ScFv gene Then corresponding restriction endonuclease digestion site was introduced into 5′and 3′ end of ScFv gene Finally, it was cloned into expression vector pCANTAB 5His and expressed in E coli HB2151 Expression proteins were purified by affinity chromatography and detected by SDS PAGE electrophoresis?Western blot and ELISA Results:Restriction endonuclease digestion and DNA sequencing proved that ScFv gene was correctly cloned into expression vector SDS PAGE electrophoresis and Western blot analysis showed that ScFv was successfully expressed in E coli HB2151 The relative molecular mass (Mr) of the expression products was 31 kD, according with its predicted Mr value And ELISA assured that expression products had antigen specific binding activity Conclusion:The successful expression of anti HAb18G ScFv gene in E coli laid a solid foundation for its further application in diagnosis and therapy of human hepatoma.

The Screening of Human Anti-Hepatoma Fd Fragments Guided with the Chemeric Light Chain of HAb18 / 中国肿瘤生物治疗杂志

Objective: To select anti-HAb18G (hepatoma associated antigen) human Fd fragments with guided selection of L chain of chimeric Fab-HAb18. Methods: The human Fd repertories genes were amplified by RT-PCR from PBMC of hepatoma patients, and cloned into the vector pComb3X with chimeric L chain to construct the human-mouse hybrid Fab phage library. HAblSGE, extracellular region of HAblSG, was used as antigen to screen. The positive clones was obtained by ELISA and FCM with p Ⅲ-fusion Fab antibody. The DNA sequences were analyzed. Results: A human-mouse Fab antibody library were constructed with 2?107 PFU. After 6 rounds panning, 7 positive clones were obtained with ELISA and FCM. And sequences of 2 clones with better affinity were same. The CHI belonged to the IgG2 class as the parent Fd, and the variable region belonged to VH3 family. Conclusions: Through the construction of the HuMFab phage antibody library and chemeric L chain-guided selection, we get the available human Fd fragments for subsequent research.

Cloning and Identification of Fd and Light Chain Genes of MAb HAb18 against Human Hepatoma / 中国肿瘤生物治疗杂志

Objective: To clone Fd and light chain genes of monoclonal antibody HAb18 against human hepatoma and verify their accuracy and liability. Methods: Total RNA was extracted from hybridoma cell line secreting MAb HAb18, and Fd and light chain genes were amplified by RT-PCR. After PCR products were ligated into pMD18T vector, positive clones were screened and DNA sequences were tested and analysed by relative softwares. Then, light chain and Fd genes were sequential cloned into phage display vector pComb3. After recombinant vector was transformed into E.coli XL1-blue, recombinant vector was rescued by helper phage M13K07 and the specificity of phages to antigen was detected by indirect ELISA. Results: The size of amplified Fd and light chain genes was separately 665 bp and 668 bp. The results of sequence analysis showed that both VL and VH contained 2 characteristic cystines and CH1 was IgG1 classes and CL was ?. ELISA result identified that expressed Fab antibody could specially bind to corresponding antigen. Conclusion: Fd and light chain genes of MAb HAb18 were successfully cloned, which lay a good foundation for constructing a diversity of engineering antibody.

Construction of human-mouse hybrid Fab phage antibody libraries / 解放军医学杂志

Objective To construct human-mouse hybrid Fab phage antibody libraries and to select target Fab genes. Methods With Fd repertoire genes of PBMC from hepatoma patients and the chemeric light chain gene of cFab-HAb18, two hybrid Fab phage antibody libraries(pComb3 and pComb3X) were constructed. The GST fusion and non-fusion HAb18GE were used as antigens to select the target antibodies with the optimized strategies of amplifying and screening. Results When pComb3 displaying library was cultivated at 37℃, the target fragments could not be cut out with the restriction endonucleases from most clones of the library, which was an indication of “recombinant-deletion”. But no “recombinant-deletion” was found in pComb3X displaying library. Because of the serious cross-reaction, the phage-display Fab ELISA could not be used for the selection of positive clones. But positive clones could be identified in the supernatant of lysate with ELISA afterbeing induced by IPTG. Conclusion Through the construction of HuMFab phage antibody libraries and the optimization of screening strategies, we get the desired HuMFab genes for subsequent research.

False positives resulted from colony PCR in rapid identification of recombinant clones from phage antibody library / 解放军医学杂志

Objective To evaluate the reliability of colony PCR in identifying the recombinant clones selected from phage antibody libraries. Methods The digested pComb3 vector and Lc fragments, Lc library plasmid, and Fd fragments were ligated successively. The ligation product was transformed into Escherichia Coli strain XL 1-blue bacilli by electroporation and thus murine Fab phage antibody library was constructed.The transformed recombinant clones selected randomly from libraries were identified simultaneously by colony PCR, plasmid PCR and restriction enzyme digestion. The identification consistency was analyzed. The interference was excluded by control PCR using the relevant constituents as template. Results The educed library content of Lc library was 1.175?10 6 CFU and the content of Fab antibody library was 1.02?10 6 CFU. Different recombinant percentages were obtained through three different identification methods (the Lc positive recombinant percentages by colony PCR, plasmid PCR and enzyme digestion were 100%, 78% and 78%, respectively; the Fd positive recombinant percentages by three methods were 90%, 66% and 66%, respectively; the dual positive recombinant (Fd and Lc insert simultaneously) percentages by three methods were 90%, 50% and 50%, respectively). A high frequency of false-positives appeared in colony PCR identification. Nonspecific amplification of control PCR was presumably induced by some intracellular components in XL 1-blue bacilli. Conclusion The identification of the recombinant clones selected from phage antibody libraries by colony PCR remains ambiguous. So it is our assertion that the traditional identification methods such as plasmid PCR or enzyme digestion are more accurate and will less false positive results.