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Objective To verify the feasibility of Cas9 microinjection in frozen-thawed pronuclear embryos, based on the model of pronuclear embryos of C57BL/6J mice by in vitro fertilization.Methods After fertilized mouse pronuclear embryos cultured in vitro, one-cell and 2-cell embryos were frozen using EFS20/40 cryopreservation tube.The next day recovered and then cultured.The recovery rate and survival rate of the one-cell and 2-cell embryos were compared.The frozen-thawed and fresh pronuclear embryos were injected with Cas9 mixture and injection buffer into the cytoplasm, and then cultivated to 2-cell embryos,and the survival rate and development rate of the 2-cell embryos were compared.Results The recovery rate of frozen-thawed one-cell embryos was 92.5%, the survival rate was 92.8%, the recovery rate of 2-cell embryos was 90.5% and the survival rate was 95.8%, showing no significant difference.Furthermore, the survival rate of fresh one-cell embryos after Cas9 injection was 92.7%, the survival rate of one-cell embryos of the blank group was 97.5%.While the survival rate of Cas9 injected frozen-thawed one-cell embryos was 82.6%, and that of the blank group was 92%,showing a significant difference between the frozen-thawed injected group and other groups(P < 0.05).The development rate of 2-cell embryos after Cas9 injection was not significantly different.Conclusions Frozen-thawed pronuclear embryos can be used for Cas9 microinjection.
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Laboratory animal science, as a supporting discipline in life science research, has been set up courses in many universities, especially in medical schools.However, due to the curriculum design was too rigidly attached to tradition, emphasis on theory and cannot adapt to the development of natural science.In this paper, aiming to enhance the teaching effect, we focus on the practice, advance with the times, combine with customized teaching reform of laboratory animal science curriculum, teaching content and teaching forms.
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Objective To investigate the characteristics of BK virus (BKV) infection in renal transplant recipients. Methods A total of 243 renal recipients from our clinic within 48 months after transplantation were enrolled as the trial group and 82 healthy people as the control group. Urine and peripheral blood samples of these two groups were harvested for urinary sediment BKV cytology by Decoy cell counting and BKV DNA by real-time PCR. Results The positive rates of urinary Decoy cell, BKV viruria and viremia were 35.4%, 36.6% and 16.9% in trial group, and 4.9%, 20.7% and 2.9% in control group, respectively. In trial group, the medians of urinary Decoy cell, urinary BKV and peripheral blood BKV were 6/10 HPF, 1.00×104 copy/ml and 6.87×103 copy/ml respectively, while in control group, they were 2/10 HPF, 1.10×104 copy/ml and 2.24×1(3 copy/ml. Compared with the healthy people, the positive rates and the levels of BKV DNA in urine and peripheral blood of recipients were significantly higher. The amount of urinary Decoy cells was positively correlated to urinary BKV load (r=0.636, P＜0.01). Conclusions BKV replication is easier to happen in renal recipients as compared to healthy people. Counting of urinary Decoy cells is convenient, useful and sensitive to evaluate BK viruria and viremia in renaltransplant recipients. BKV DNA detection in urine and peripheral blood can be used to screen the evidence of BK reaction in order to prevent irreversible graft damage by BKV.[ Key words ] Kidney transplantation; BK virus; Kidney diseases; Decoy cells
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Objective To study the expression of tumor type M2 pyruvate kinase(tumor M2-PK) in nonsmall cell lung cancer (NSCLC) and their correlation with treatment response and prognosis.Methods The concentration of tumor M2-PK in plasma was detected by ELISA in 106 healthy controls and 83 NSCLC patients.The patients were followed for 24 months.Results The patients after surgical operation showed marked reduction in plasma tumor M2-PK level(13.5 U/ml vs 25.4 U/ml,P<O.05).In the 15 patients with tumor remission the tumor M2-PK level(11.5 U/ml) was significantly lower than that(22.6 U/ml) before treatment(P<0.05).In the 20 patients with progressive disease the tumor M2-PK level(60.8 U/ml) was significantly higher than that(23.7 U/ml) of pretreatment(P<0.05).The tumor M2-PK concentration did not differ signigicantly before and after treatment in 17 patients with stable disease.The positive rates of tumor M2-PK decreased significantly after operation,but there was no significant change after chemotherapy.In the follow-up,the rate of relapse and metastasis was higher in patients Conclusions Plasma tumor M2-PK might be a useful marker in the detection of tumor mierometastases in NSCLC,and in evaluating treatment response and prognosis.
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might be related to the growth and metastasis of HCC.
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Objective To observe the changes of soluble interleukin-2 receptor(slL-2R) and high sensitivity C-reactive (hs-CRP) protein in renal allograft recipients and its relationship with rejective response of renal allograft. Methods The concentration of sIL-2R and hs-CRP were detected in 91 patients with renal allograft recipients and 100 health controls. CRP was measured by particle enhanced immuo-turbidimetric method, sIL-2R was detected by ELISA. Results The concentration of sIL-2R and hs-CRP in the recipients with acute rejec-tion was significantly higher than those without graft rejection and health controls. The concentration of hs-CRP in the recipients with chronic rejection was higher than those without graft rejection and health controls. There was no evidence of significant changes in the concentration of sIL-2R and hs-CRP between the health control group and the recipients without graft rejection. Conclusion slL-2R and hs-CRP might play an important role in graft rejection. The measurement of sIL-2R and hs-CRP is important for the diagnosis of graft rejection.
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Objective To evaluate the practical value of Serology, PCR-SSP and PCR-SSOP for HLA-B typing. Methods A total of 30 samples, the blood of patients and donors waiting kidney transplantation, were used in the study. HLA-B typing was performed by one-step monoclonal antibody typing, micro PCR-SSP typing and PCR-SSOP reverse hybridisation. Results All samples were successfully typed by three methods. The difference between serological and PCR-SSP typing was 13%. The difference between PCR-SSOP and PCR-SSP typing was 3%. Conclusion Serology is high discrepancy rate and low-resolution, but cheap, simple and rapid. PCR-SSP and PCR-SSOP typing are high specific and accuracy. SSP is suitable for several samples, and SSOP is for lots of samples simultaneously although it needed long time.
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Objective To study the distribution and tumor targeting property of anti-human prostate specific membrane antigen(PSMA) monoclonal antibody Ed-5 labeled with ~ 131 I in nude mice bearing human prostate cancer. Methods The nude mice xenografted models of human prostate cancer were established. Anti-human PSMA monoclonal antibody Ed-5 was labeled with ~ 131 I. ~ 131 I-Ed-5 was injected through the tail vein of nude mice bearing human prostate cancer. r-ray scintigraphy was performed at 24,48,96 and 144 hours after injection. And then the mouse were killed, the radioactivity ratio of tumor and non-tumor (T/NT), percentage of injected dosage of every gram tissue (%ID/g) were measured, and the bio-distribution of the labeled antibody was determined. Results The tumor imaging was clearly visualized at 96 hours after ~ 131 I-Ed-5 injection, and the tumor brim was clearer along with time prolonging. Radioactivity assembled in the tumors 24 to 144 hours after ~ 131 I-Ed-5 injection. The tumor's %ID/g at 96 hours after injection was 32.30, the radioactivity ratio of tumor and various tissues reached a maximum, and that of tumor/muscle was 7.08. Conclusion Ed-5 exhibited excellent immunoreactivity and tumor targeting property, and had application potential in targeting diagnosis and therapy of prostate cancer.
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Objective To investigate the significance of enzyme-linked immunosorbent assay (ELISA) in cross-match test of donor-recipient before organ transplantation.Methods HLA glycoproteins were prepared by solubilizing the lymphocytes of donor with a non-ionic detergent, and bond to the monoclonal antibody specific for HLA class Ⅰ or Ⅱ immobilized in the ELISA tray. ELISA with addition of recipients' serum and complement-dependent cytotoxicity (CDC) were compared.Results Cross-match test was performed by two methods for lymphocytes of 40 donors and sera of 72 recipients. All samples were successfully tested. The results of one pair of donor-recipient cross-match test by two methods were different.Conclusion ELISA for cross-match test is simple, convenient and time-saving, but more sensitive and specific than CDC.
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Objective:Preformed anti-human leukocyte antigen(HLA)specific antibodies are a major risk for antibody-mediated rejection.The aim of this study is to detect and analyze anti-HLA specific antibodies in sera of renal transplant candidates for evaluating the status of presensitization.Methods:A total of 3 500 patients awaiting renal transplantation in our hospital from 1998 to 2007 were included in the study.Panel reactive antibody(PRA)in sera of 694 candidates was detected by complement-dependent cytotoxicity(CDC)before March 2000,then the sera of other 2 806 recipients were screened by enzyme-linked immune absorbent assay(ELISA)alternatively.The polymorphism,specificity and relevant factors of anti-HLA antibodies were analyzed.Results:Only IgG type of antibodies specific to HLA classⅠcould be detected by CDC,whereas both anti-HLA classⅠand classⅡ of IgG class could be found out by ELISA.Lower PRA positive rate by CDC(8%)was shown when compared to that by ELISA(17%).Furthermore,multiple types of specific antibodies against HLA-A(20),B(37),CW(8),DR(14)and DQ(7)were determined by ELISA.Some types of the antibodies presented higher frequencies,such as anti-HLA-A2,24,68,23 and 32;B27,56,57 and 7;DR7,4,9,13,17 and 12;CW1,2,6,4 and 8.These high frequenies of anti-HLA antibodies were somewhat different from the distribution of HLA antigens in South China population.There were significantly different positive rates of anti-HLA antibodies between the male and the female,as the male were sensitized mainly through blood infusion and the female were sensitized by either blood infusion or pregnancy.Conclusion:Specific antibodies against HLA can be detected out by ELISA accurately,whereby to find high freguencies of the antibodies and to avoid donor-recipient mismatching at HLA-loci.Detection of preformed anti-HLA and reducion of HLA-mismatched blood infusion to reduce production of anti-HLA antibodies may be the valuable pathway to improve graft survival.