目的 探讨腺病毒载体(Ad-DDAH2)转染大鼠脂肪干细胞(ADSCs)的可行性.方法 构建Ad-DDAH2-GFP质粒.原代培养大鼠ADSCs,流式细胞术检测免疫表型CD29、CD45、CD90表达情况.以不同滴度的Ad-DDAH2-GFP转染ADSCs,用荧光显微镜计数转染效率及细胞形态.通过RT-PCR及Western Blot检测DDAH2在ADSCs中的表达情况.结果 Ad-DDAH2-GFP重组腺病毒载体构建、包装成功,且病毒滴度达到2E+10PFU/ML.第4代ADSCs生长曲线呈现"S"形.细胞免疫表型CD29、CD90阳性率分别为(95.83±0.53)％、(91.32±0.27)％,CD45阳性率为(1.59±0.06)％,符合ADSCs免疫表型特征.当病毒感染复数值为80、100时,转染效率均可达到90％以上.但病毒感染复数值为100时,ADSCs出现明显细胞病理现象,因此病毒感染复数值为80被视为最佳感染复数,RT-PCR及Western Blot鉴定可见阳性扩增条带.结论 Ad-DDAH2-GFP重组腺病毒载体构建成功,可以高效转染大鼠ADSCs.DDAH2在mRNA及蛋白稳定长期表达,为后续体内实验奠定了实验基础.

慢性前列腺炎是较为常见的男科疾病,患病率在10%左右 [1] ,据统计约50%的男性在一生中的不同时期会出现前列腺炎相关的症状 [2] 。研究表明,慢性前列腺炎与男性不育症关系密切,其中Ⅲ型前列腺炎（慢性前列腺炎/慢性骨盆疼痛综合症）是其

OBJECTIVE:To investigate the role of pH2AX in the reversibility of mouse testicular reproductive function impaired by single heat stress.METHODS:Twenty-four C57 male mice were randomly divided into heat stress and control groups and immersed in water at 43℃ and 25℃, respectively, for 15 minutes. At 1, 7, and 14 days of heat exposure, all the mice were sacrificed and their testis tissues collected for determining the apoptosis of the germ cells by TUNEL and measuring the expression level of the pH2AX protein by immunohistochemistry and Western blot.RESULTS:The highest percentage of apoptotic cells were found in the seminiferous tubules of the mice in the heat stress group on the 1st day of the exposure and almost no apoptosis was observed at 7 and 14 days. The pH2AX protein was expressed in the nuclei of the basement membrane of adjacent seminiferous tubules. Compared with the control group, the expression of pH2AX was significantly increased on the 1st day of exposure (0.47 ± 0.02 vs 1.61 ± 0.04, P <0.01), then decreased at 7 days (0.85 ± 0.03) in comparison with that on the 1st day (P <0.01), and again elevated at 14 days (1.72 ± 0.02) as compared with either those at 1 and 7 days (P <0.01) or that of the control (P <0.01).CONCLUSIONS:Heat stress causes dynamic changes of the pH2AX expression in the testis of the mouse, which are associated with heat stress-induced proliferation and division of the testicular spermatogenic cells.

目的:小鼠弓状核损毁是否影响胃组织和血浆nesfatin-1水平.方法:新生健康昆明小白鼠.谷氨酸单钠(MSG)处理组小鼠出生后1、3、5、7、9d于颈部皮下注射MSG.取胃组织行RT-PCR、免疫印迹,检测nesfatin-1 mRNA及蛋白质水平,分别断头取脑,冷冻切片观察弓状核损毁情况.结果:与生理盐水组小鼠相比,MSG组小鼠弓状核甲酚紫染色神经元明显减少,体质量明显增加;禁食48 h后胃黏膜组织NUCB2 mRNA及蛋白表达明显低于禁食前,血浆nesfatin-1水平明显低于禁食前;MSG组小鼠与生理盐水组小鼠比较,在禁食前后,胃黏膜组织NUCB2 mRNA和蛋白表达水平无明显改变,血浆nesfatin-1水平无明显改变.结论:弓状核损毁并没有影响胃黏膜NUCB2/nesfatin-1的合成、分泌以及血浆nesfatin-1水平.

Objective To design, synthesize screen small interfering RNA (siRNA) targeting to Casitas B-cell lineage lymphoma-b (cbl-b).Methods Four pairs of 21 nucleotide siRNAs directed to Cbl-b mRNA were designed and synthesized by utilizing RNA design software to simulate secondary structure of cbl-b mRNA in mice. These siRNAs were respectively transfected into lymphocytes in 96 shadows mask by oligofectamine package, and untreated and unspecific siRNA-transfected lymphocytes served as controls. The expression of cbl-b protein was detected by Western blotting.Results When the work concentration of siRNA was 100 nmol/L, transfection efficiency of lymphocytes was highest, up to (87.48±1.94)% and the mean fluorescence intensity was strongest, up to 33.09±1.77. Compared with bland controls, the expression of cbl-b protein level was markedly down-regulated in siRNA-transfected lymphocytes. The inhibitory rate of the siRNA of the target-4 was highest, up to 85%. The expression of cbl-b protein in unspecific siRNA-transfected lymphocytes had no significant changes.Conclusion siRNA-4, which can highly effectively inhibit protein expression of cbl-b gene, was screened successfully, and its inhibition effect can maintain near 48 h. It is hopeful that the cbl-b siRNA will activate lymphocytes directly by cbl-b gene silencing, and kill tumor by activate immunization.