Knockout and identification of the surface antigen 43 gene in escherichia Coli JM109 / 重庆医学

Objective To knockout and identify the Antigen 43 (Ag43) in the Escherichia Coli JM109 .Methods Mutation group Ⅱ introns RNA protein complexes (RNP) gene sequence was obtained by Sigma Company′s TargeTron Gene Knockout Sys‐tem and Ag43 gene specific designed PCR primers amplification ,then ,to acquired Ag43 specific recombinant RNP plasmid pACD4K‐Ag4 ,this gene sequence was inserted into the plasmid pACD4K‐C of RNA′s expression .Finally ,pEGFP‐Ag43 was trans‐formed into JM109 and inserted the group Ⅱ intron into the Ag43′s locus by IPTG inducing expression .Results The best insertion locus was between 1 812 and 1 913 .Through the agarose electrophoresis gel ,the RNP gene sequence was consistent with the expec‐ted value (350 bp) .The pEGFP‐Ag43 vector was correctly constructed which was proofed by endonuclease Nhe Ⅰ and Hind ⅡI di‐gestion as predicted products (3 646 and 4 029 bp;7 000 and 550 bp ,respectively ) .The PCR and gene sequence results indicated that the group Ⅱ intron was inserted into the locus between 1 812 and 1 913 in the Ag43 gene .Conclusion Successful knockout of the Ag43 in Escherichia Coli JM109 found basis to further study the Ag43′s function and regard the coli as host bacteria of Ag43 chimeric protein recombinant .

Isolation and identification of multidrug-resistant Staphylococcus haemolyticus from a laboratory-breeding mouse

OBJECTIVE@#To analysis and identify a bacterium strain isolated from laboratory breeding mouse far away from a hospital.@*METHODS@#Phenotype of the isolate was investigated by conventional microbiological methods, including Gram-staining, colony morphology, tests for haemolysis, catalase, coagulase, and antimicrobial susceptibility test. The mecA and 16S rRNA genes were amplified by the polymerase chain reaction (PCR) and sequenced. The base sequence of the PCR product was compared with known 16S rRNA gene sequences in the GenBank database by phylogenetic analysis and multiple sequence alignment.@*RESULTS@#The isolate in this study was a gram positive, coagulase negative, and catalase positive coccus. The isolate was resistant to oxacillin, methicillin, penicillin, ampicillin, cefazolin, ciprofloxacin erythromycin, et al. PCR results indicated that the isolate was mecA gene positive and its 16S rRNA was 1 465 bp. Phylogenetic analysis of the resultant 16S rRNA indicated the isolate belonged to genus Saphylococcus, and multiple sequence alignment showed that the isolate was Saphylococcus haemolyticus with only one base difference from the corresponding 16S rRNA deposited in the GenBank.@*CONCLUSIONS@#16S rRNA gene sequencing is a suitable technique for non-specialist researchers. Laboratory animals are possible sources of lethal pathogens, and researchers must adapt protective measures when they manipulate animals.

Inhibitory effect of GnRHII and GnRH Ia on the stromal cell proliferation from endometriosis patients / 中南大学学报(医学版)

OBJECTIVE@#To investigate the inhibitory effect of gonadotropin-releasing hormone II(GnRHII) and gonadotropin-releasing hormone I agonist (GnRH Ia) on the proliferation of endometrial stromal cells in vitro from endometriosis patients.@*METHODS@#Different concentrations of GnRHII or GnRH Ia were added into the cultured endometrial stromal cells in vitro to detect the cell proliferation inhibition by MTT test.@*RESULTS@#The inhibitory rate of GnRHII or GnRH Ia on eutopic and ectopic endometrial stromal cells in vitro was both dose- and time-dependent (P<0.05). Effect of GnRHII or GnRH Ia on the inhibitory rate of ectopic endometrial stromal cells was significantly higher than that of eutopic (P<0.05). GnRH II had a higher inhibitory rate on the endometrial stromal cells in vitro than did GnRH Ia (P<0.05).@*CONCLUSION@#GnRHII has more antiproliferative effect on endometrial stromal cells than GnRH Ia in vitro, especially on ectopic endometrial stromal cells, suggesting that GnRHII may be more effective than GnRH Ia on endometriosis.

Activin and activin receptors in related gynecologic and obstetric diseases / 中南大学学报(医学版)

Activin is a member of the transforming growth factor-beta (TGF-beta) superfamily that result from the assembly of disulphide-linked betaA and betaB subunits. Activin receptors are transmembrane proteins and activin fulfils the biological function through the signal transduction of the receptor system. In recent years, many studies have suggested that activins have wide biological activities. It is the basic medium in regulating histiocytic function and plays a role in maintaining the normal function of cells. Moreover, abnormal expression of activin in the tissues of many gynecologic and obstetric diseases, such as epithelial ovarian tumor, endometrial carcinoma, pre-eclampsia, polycystic ovary syndrome, endometriosis and so on affects the development of these diseases.

Effect of GnRH Ⅱ and GnRH Ⅰ on secretion of VEGF by eutopic and ectopic endometrial stromal cells of endometriosis patients / 中南大学学报(医学版)

Objective To determine the effect of GnRH Ⅰ and GnRH Ⅱ on the secretion of VEGF by eutopic and ectopic endometrial stromal cells cultured in vitro, and to provide theoretical basis for exploring new treatments for endometriosis (EMs).Methods Eutopic and ectopic endometrium stromal cells cultured in vitro were treated with different concentrations of GnRH Ⅱ and a GnRH I(goserelin), and a control group was not treated by GnRH Ⅱ and GnRH Ⅰ. Enzyme linked immunosorbent assay (ELISA) was used to measure the content of vascular endothelial growth factor (VEGF) protein in the medium of the above 2 groups.Results (1)There was no difference in the VEGF protein secreted by eutopic and ectopic stromal cells in the medium after being cultured in vitro for 48 h (P＞0.05).(2)10-10, 10-8, and 10-6mol/L GnRH Ⅱ dose-dependently reduced VEGF protein secreted by endometrial stromal cells (P＜0.05),and the inhibition effect was stronger than that of GnRH Ⅰ (P＜0.05).(3)The inhibition effect of GnRH Ⅱ on VEGF in ectopic stromal cells was stronger than that of eutopic stromal cells (P＜0.05).Conclusion (1)Ectopic stromal cells cultured in vitro can secrete VEGF,which has no difference from the eutopic stromal cells, and which may play an important role in the formation and development of EMs.(2)GnRH Ⅱ can dose-dependently reduce VEGF protein secreted by ectopic and eutopic endometrial stromal cells cultured in vitro,and the inhibition effect is stronger than that of GnRH Ⅰ, providing theoretical basis for exploring new treatments for EMs.

Anti-tumor angiogenesis with a recombinant ag43/FGFR1 chimeric protein as a model antigen / 华中科技大学学报（医学）（英德文版）

In order to investigate the anti-tumor angiogenesis activity with a recombinant Ag43/FGFR1 chimeric protein (AF) vaccine in a mouse H22 hepatoma model, tumor volume and survival rate of the mice were studied at a 3-day interval. Microvessel density (MVD) was detected by immunohistochemistry. The endothelial deposition of autoantibodies within tumor tissues was examined by immunofluorescent staining, and anti-FGFR1 antibody-producing B cells (APBCs) were tested by enzyme-linked immunospot (ELISPOT) assay. Compared with the three control groups, the tumor volume was significantly decreased and the survival time was significantly prolonged in AF-immunized group (P<0.05). The number of APBCs in AF-immunized mice (129.6+/-10.9) was more than in controls [6.2+/-1.1 (FGFR1), 6.0+/-1.2 (Ag43) and 5.2+/-1.4 (NS), P<0.01]. Moreover, the endothelial deposition of autoantibodies was found in tumor tissues from AF-immunized mice, but not in control groups. MVD in AF-immunized group was significantly lower than in FGFR1-immunized group, Ag43-immunized group and NS group (10.3+/-3.1 vs 39.4+/-8.6 vs 42.3+/-9.8 and 43.6+/-10.6, P<0.01). These findings demonstrated that the AF protein vaccine effectively inhibited tumor angiogenesis and growth via production of autoantibodies against self-FGFR1.

Effect of GnRH-II on VEGF secreted by stromal cells from endometrium of endometriosis / 中南大学学报(医学版)

OBJECTIVE@#To inspect the effect of gonadotropin-releasing hormonel II (GnRH-II)on the secretion of VEGF by eutopic and ectopic endometrial stromal cells cultured in vitro.@*METHODS@#Eutopic and ectopic stromal cells cultured in vitro were treated with different concentrations (1x10(-10) - 1x10(-6) mol/L) of GnRH-II,and the control group was not treated by GnRH-II.Enzyme-linked immunosorbent assay (ELISA) was used to measure the VEGF protein in the medium of the above 2 groups.@*RESULTS@#There was no difference between the VEGF protein expressed by eutopic and ectopic stromal cells in the medium after culturing in vitro for 48 hours (P>0.05). The 1x10(-10) - 1x10(-6) mol/L GnRH-II could dose-dependently reduce VEGF protein secreted by endometrial stromal cells (P<0.01), and the inhibition to ectopic endometrial stromal cells was stronger than that to eutopic endometrial stromal cells (P<0.01).@*CONCLUSION@#Ectopic stromal cells cultured in vitro can secrete VEGF, and so can eutopic stromal cells. This may play an important role in the formation and development of endometriosis. GnRH-II can reduce VEGF protein secreted by ectopic endometrial stromal cells cultured in vitro, and its inhibition is stronger than that of eutopic endometrial stromal cells.

Expression and significance of KISS-1 in ectopic endometrium in patients with endometriosis / 中国基层医药

Objective To study the expression of KISS-1 in normal endometrium and in ectopic endometri-um patients with endometriosis. Methods The expression of KISS-1 in 55 cases of normal endometrium as well in 46 cases of ectopie endometriurn with endometriosis were detected by immunohistochemicai EliVision plus method. Results The positive expression rates of KISS-1 in ectopic endometriotic tissues and in normal endometri-urn were 30.43 % and 63.64 % respectively ,and the positive expression rates in ectopic endometriotic tissues signifi-cantly lower than those in the normal endometrium( P< 0.01, P < 0.05). Conclusion The decreased expression of KISS-1 may be related to the mechanism of endometriosis.

Anti-angiogeneic target therapy for cancer with vaccine based on the recombinant chicken FGFR-1 in tumor-bearing mice / 华中科技大学学报（医学）（英德文版）

To explore the anti-tumor effect of immunotherapy with recombinant protein vaccine based on FGFR-1 of chicken (cFR-1) in a mouse Meth A fibrosarcoma model, tumor volume and survival rate of the mice were observed at a 3-day interval. Microvessel density (MVD) was detected by immunohistochemistry. Auto-antibodies against self-FGFR-1 were detected by Western blotting and ELISA, respectively. The anti-FGFR-1 antibody-producing B cells (APBCs) were detected by enzyme-linked immunospot (ELISPOT) assay. Eighteen days after inoculation of tumor cells, the tumor volume was significantly smaller in cFR-1-immunized group than in mouse FGFR-1 (mFR-1) immunized group and normal saline (NS) control group (P<0.05), and the survival time was significantly longer in cFR-1-immunized group than in the control groups (P<0.01). MVD was significantly lower in cFR-1-immunized group than in mFR-1-immunized group and NS group (16.8+/-5.6 vs 64.6+/-1.8 and 59.6+/-8.7, P<0.01). Antibodies against self-FGFR-1 were found in mFR-1-immunized group, the major antibody subclasses were IgG1 and IgG2b. Compared with the two control groups, the numbers of APBCs in cFR-1-immunized group were significantly increased (P<0.01) These results demonstrated that the cFR-1-related anti-angiogenesis protein vaccine could induce the production of auto-antibodies against self-FGFR-1, which futher inhibit angiogenesis and growth of solid tumor.

Anti-angiogeneic Target Therapy for Cancer with Vaccine Based on the Recombinant Chicken FGFR-1 in Tumor-bearing Mice / 华中科技大学学报（医学）（英德文版）

To explore the anti-tumor effect of immunotherapy with recombinant protein vaccine based on FGFR-1 of chicken (cFR-1) in a mouse Meth A fibrosarcoma model, tumor volume and survival rate of the mice were observed at a 3-day interval. Microvessel density (MVD) was detected by immunohistochemistry. Auto-antibodies against self-FGFR-l were detected by Western blotting and ELISA, respectively. The anti-FGFR-1 antibody-producing B cells (APBCs) were detected by enzyme-linked immunospot (ELISPOT) assay. Eighteen days after inoculation of tumor cells, the tumor volume was significantly smaller in cFR-l-immunized group than in mouse FGFR-1 (mFR-1) immunized group and normal saline (NS) control group (P＜0.05), and the survival time was significantly longer in cFR-l-immunized group than in the control groups (P＜0.01). MVD was significantly lower in cFR-l-immunized group than in mFR-l-immunized group and NS group (16.8 ±5.6 vs 64.6±1.8and 59.6±8.7, P＜0.01). Antibodies against self-FGFR-1 were found in mFR-l-immunized group, the major antibody subclasses were IgG1 and IgG2b. Compared with the two control groups, the numbers of APBCs in cFR-l-immunized group were significantly increased (P＜0.01) These results demonstrated that the cFR-1-related anti-angiogenesis protein vaccine could induce the production of auto-antibodies against self-FGFR-1, which futher inhibit angiogenesis and growth of solid tumor.

The analysis of effect of preoperative chemotherapy on bulky cervical cancer by internal illiac arterial infusion / 肿瘤研究与临床

Objective To investigate the therapeutic effect of arterial interventional chemotherapy on bulky cervical cancer. Methods One hundred and twenty-six patients with bulky cervical cancer were randomly divided into two groups. Arterial interventional chemotherapy group(n =74, C gronp), and radiotherapy group(n =52 R group). Patients in C group underwent internal iliac arterial infusion chemotherapy by using Seldinger technique. The chemotherapy regimens of cervical squamous cell carcinoma were prescribed including cisplatin and BLM, and cervical adenocarcinoma were prescribed including cisplatin and ADM and VCR. Patients in R group were only given radiotherapy Ir192 high-dose rate intracavitary radiotherapy was performed with A point dose at 24 Gy. Both groups of patients were followed up after two weeks. Results The tumor regression rate of C group was 93.24 %, significantly higher than 71.15 % in R group(P 0.05). The 3-year recurrence rate between the two groups had obvious difference(P 0.05). Conclusion Neoadjuvant chemotherapy can effectively reduce tumor volume, and postoperative 3-year recurrence rate, increases surgery rate on bulky cervical cancer. But the effect on long-term survival rate needs to be evaluated further through long-term follow-up.

Vaccination with a Recombinant Chicken FGFR-1 Bypasses Immunological Tolerance against Self-FGFR-1 in Mice / 华中科技大学学报（医学）（英德文版）

The possibility that a recombinant protein vaccine based on xenogeneic homologous FGFR-1 of chicken induces production of autoantibodies against self-FGFR-1 in BALB/c mice was examined by using ELISA, Western blot analysis and ELISPOT assay respectively. Autoantibodies against mouse FGFR-1 were identified by Western blot analysis and ELISA. Compared with the two control groups, the number of APBCs, which were detected by ELISPOT assay, was significantly increased in the spleens of mice immunized with cFR1 (P＜0.05). IgG1 and IgG2b, which were detected by ELISA, were the major subclasses and were substantially increased in response to chicken FGFR-1 when compared with control group. The recombinant chicken FGFR-1 protein used as a vaccine can induce autoantibodies against self-FGFR-1 in mice and provide a basis for the active immunotherapy of tumor angiogenesis.

Vaccination with a recombinant chicken FGFR-1 bypasses immunological tolerance against self-FGFR-1 in mice / 华中科技大学学报（医学）（英德文版）

The possibility that a recombinant protein vaccine based on xenogeneic homologous FGFR-1 of chicken induces production of autoantibodies against self-FGFR-1 in BALB/c mice was examined by using ELISA, Western blot analysis and ELISPOT assay respectively. Autoantibodies against mouse FGFR-1 were identified by Western blot analysis and ELISA. Compared with the two control groups, the number of APBCs, which were detected by ELISPOT assay, was significantly increased in the spleens of mice immunized with cFR1 (P < 0.05). IgG1 and IgG2b, which were detected by ELISA, were the major subclasses and were substantially increased in response to chicken FGFR-1 when compared with control group. The recombinant chicken FGFR-1 protein used as a vaccine can induce autoantibodies against self-FGFR-1 in mice and provide a basis for the active immunotherapy of tumor angiogenesis.