目的 探讨干扰长链非编码RNA STX18-AS1(long non-coding RNA,LncRNA STX18-AS1)是否通过上调微小RNA-204(miR-204)的表达从而影响心肌细胞缺氧损伤.方法 体外培养心肌细胞H9C2,采用二氯化钴(CoCl2)处理心肌细胞建立细胞缺氧损伤模型,采用qRT-PCR检测CoCl2处理不同时间点STX18-AS1、miR-204 的表达量;实验设置对照组、模型组、si-NC组、si-STX18-AS1组、miR-NC组、miR-204组.采用甲基噻唑基四唑(MTT)检测细胞增殖活性;流式细胞术与TUNEL检测细胞凋亡率;采用生化试剂盒检测乳酸脱氢酶(LDH)活性、丙二醛(MDA)水平、超氧化物歧化酶(SOD)活性、过氧化氢酶(CAT)活性;双荧光素酶报告实验验证STX18-AS1、miR-204的靶向关系.结果 与对照组比较,在CoCl2处理6 h、12 h、18 h、24 h时模型组和si-NC组STX18-AS1表达水平升高(P＜0.01),miR-204表达水平降低(P＜0.01);与模型组、si-NC组比较,在CoCl2处理6h、12h、18h、24 h时si-STX18-AS1组STX18-AS1的表达水平降低(P＜0.01),miR-204的表达水平升高(P＜0.01);与对照组比,模型组、si-NC组细胞增殖活性降低(P＜0.01),细胞凋亡率及LDH活力、MDA的水平升高(P＜0.01),SOD、CAT的活力降低(P＜0.01);与模型组、si-NC组比较,si-STX18-AS1组细胞增殖活性升高(P＜0.01),细胞凋亡率及LDH活力、MDA的水平降低(P＜0.01),SOD、CAT的活力升高(P＜0.01);转染miR-204 mimics对心肌细胞增殖活性、凋亡及氧化应激的作用与转染si-STX18-AS1的作用相同;双荧光素酶报告实验证实STX18-AS1可靶向结合miR-204.结论 干扰STX18-AS1表达可能通过上调miR-204,抑制细胞凋亡、促进增殖,减轻CoCl2 诱导的心肌细胞氧化损伤.

目的 探讨四味地黄醇提取物对高糖作用的心肌细胞凋亡的影响及作用机制.方法 实验设置对照组、模型组、模型+四味地黄醇提取物-L组、模型+四味地黄醇提取物-M组、模型+四味地黄醇提取物-H组、模型+miR-NC组、模型+miR-26b组、模型+四味地黄醇提取物-M+anti-miR-NC组、模型+四味地黄醇提取物-M+anti-miR-26b组.流式细胞术检测细胞凋亡;乳酸脱氢酶(lactate dehydrogenase,LDH)试剂盒、超氧化物歧化酶(superoxide dismutase,SOD)试剂盒、丙二醛(malondialdehyde,MDA)试剂盒分别检测LDH、SOD活性及MDA含量;实时荧光定量PCR(RT-qPCR)检测miR-26b和MAPK mRNA表达水平;荧光素酶报告实验检测miR-26b和MAPK的靶向关系.结果 与对照组相比,模型组心肌细胞凋亡率显著升高,MDA含量、LDH活性显著升高,SOD活性显著降低,miR-26b表达水平显著降低,MAPK mRNA表达水平显著升高(P<0.05).低、中、高浓度四味地黄醇提取物处理可降低细胞凋亡率和MDA含量、LDH活性,提高SOD活性,提高miR-26b表达水平,降低MAPK mRNA表达水平(P<0.05).过表达miR-26b抑制高糖诱导的心肌细胞凋亡和氧化应激的产生.抑制miR-26b逆转了四味地黄醇提取物对高糖作用的细胞H9C2凋亡和氧化应激的抑制作用.miR-26b靶向调控MAPK.结论 四味地黄醇提取物可抑制高糖作用的细胞H9C2凋亡和氧化应激,其机制可能与miR-26b及MAPK表达有关.

Whole cell lysate of mouse hepatoma(H22) was prepared and processed to the whole tumor cell vaccine H22-DT-M2-OK432(abbreviated as HDTMOK) by chemical coupling a mixture of DT and microbial HSP70 peptide epitope 407-426 and addition of OK432 as antigen.According to the results of therapeutic immune,the immune response stimulated by HDTMOK had effectively inhibited the growth of H22.Compared with that of the PBS group,the average weight and size of the tumor had both significantly reduced(P<0.05).In order to enhance the anti-tumor effect of the vaccines,immunostimulating complex(ISCOM) were prepared on the basis of HDTMOK.Therapeutic immune results showed that the mean weight and size of excised tumors significantly reduced compared with that of the PBS group(P<0.01).ISCOM also effectively retarded the angiogenesis of intradermal tumor model(P<0.01).Meanwhile,the anti-tumor effect was also strengthened in comparison with HDTMOK(P<0.05).

Cancer cell vaccine-based immunotherapy has received increasing interest in many clinical trials involving patients with breast cancer. Combining with appropriate adjuvants can enhance the weak immunogenic properties of tumor cell lysates (TCL). In this study, diphtheria toxin (DT) and two tandem repeats of mycobacterial heat shock protein 70 (mHSP70) fragment 407-426 (M2) were conjugated to TCL with glutaraldehyde, and the constructed cancer cell vaccine was named DT-TCL-M2. Subcutaneous injection of DT-TCL-M2 in mice effectively elicited tumor-specific polyclonal immune responses, including humoral and cellular immune responses. High levels of antibodies against TCL were detected in the serum of immunized mice with ELISA and verified with Western blot analyses. The splenocytes from immunized mice showed potent cytotoxicity on Ehrlich ascites carcinoma cells. Moreover, the protective antitumor immunity induced by DT-TCL-M2 inhibited tumor growth in a mouse breast tumor model. DT-TCL-M2 also attenuated tumor-induced angiogenesis and slowed tumor growth in a mouse intradermal tumor model. These findings demonstrate that TCL conjugated with appropriate adjuvants induced effective antitumor immunity in vivo. Improvements in potency could further make cancer cell vaccines a useful and safe method for preventing cancer recurrence after resection.

To express staphylococcal protein A(SPA) in E.coli expression system at a high level,L-AsPsII signal peptide was added ahead of protein A gene containing E,D,A,B,C domains.Synthetic recombinant protein A gene,of which the rare codon was substituted,was inserted into pET28a and then transformed into E.coli BL-21(DE3).The recombinant pET28a-Pro A plasmid was expressed at a high level in BL21(DE3) by lactose induction,more than 80% of the recombinant protein A was secreted into the culture medium and the expression level was up to 375 mg/L.The culture medium containing protein A was ultrafiltrated and purified by DEAE.Using the one step purification,the purity of target protein was up to 95% and there was only one band at 32 ku by SDS-PAGE.The recombinant protein A had a high activity detected by ELISA,even boiled for 10 min,and it also had high affinity with different IgGs from mouse,rabbit and goat.

To make tumor vaccine B16F10-DT-M2-OK432,the freeze-thaw method was adopted to obtain B16F10 tumor cell lysate.Diphtheria toxin(DT),OK432 or M2 which was two tandem repeats of sequence 407-426 of microbial HSP70 were selected as carrier and adjuvants respectively.The C57BL/6 mice were immunized with B16F10-DT-M2-OK432 in order to explore whether the tumor cell vaccine can effectively inhibit B16F10 melanoma in tumor-bearing mice and to study the mechanism of B16F10-DT-M2-OK432.After the last immunization,humoral immune and cellular immune response were detected.High titer of anti-B16F10 tumor cell lysate antibody was detected in immunized mice sera by ELISA.Splenic lymophocyte proliferation assay results showed that the proliferation activity of splenocytes from mice immunized with B16F10-DT-M2-OK432 vaccine was significantly higher.The results of prophylactic/therapeutic experiment showed that B16F10-DT-M2-OK432 immunization significantly inhibited the growth of B16F10 tumor in mice.The tumor latency was prolonged and the average tumor weight in the mice immunized with B16F10-DT-M2-OK432 was significantly lower than that of mice injected with PBS(P0.05).Meanwhile,it also attenuated tumor-induced angiogenesis in intradermal tumor model mice(P0.01).B16F10-DT-M2-OK432 vaccine can effectively suppress the growth of B16F10 melanoma in mice.

AIM: To evaluate the transfection efficiency and cytotoxicity of polyamine cationic liposome(PCL)prepared by ourselves. METHODS: PCL with polyamine cationic lipids TC-Chol and neutral phospholipid DOPE at a molar ratio of 3∶1 was prepared,and PCL or Lipofectamine 2000 (as control) mediated by the plasmid PIRES2-EGFP was transfected into HeLa cells or Hep2 cells,and the expression of EGFP was determined by fluorescence microscope.The survival fraction was determined by MTT to evaluate the transfection efficiency and cytotoxicity after transfection. RESULTS: The transfection efficiency of PCL prepared with TC-Chol and DOPE at a molar ratio of 3∶1 was a few lower than that of Lipofectamine 2000,but the cytotoxicity of the former was less than the latter. CONCLUSION: PCL has a sound transfection activity and less cytotoxicity compared with Lipofectamine 2000.It is shown to be promising for gene transfection and gene therapy.

[Objective] To prepare polyamine cationic liposome(PCL), and transfect it into mammlian cell to assess transfection efficiency and cytotoxicity, and to optimize the condition of transfection. [Methods] PCL with polyamine cationic lipids TC-Chol and neutral phospholipid DOPE at a certain molar ratio was prepared, the plasmid PIRES2-EGFP which mediated PCL was transfected into Hela cells, and the expression of EGFP was determined by fluorescence microscope. The survival fraction rate was determined by MTT to evaluate the cytotoxicity post-transfection for optimization of the transfection condition of mammalian cell. [Results] PCL mediated transfection could enhance the transfection efficiency, at the mass ratio of 3～4∶1 and it was more efficient and less cytotoxic. [Conclusion] PCL prepared with TC-Chol and DOPE can enhance the transfection efficiency, and satisfactory transfection activity and less cytotoxicity is observed under certain condition. It provides laboratary data for gene transfection and gene therapy.