沉默PTEN基因对PC-3细胞增殖和凋亡的影响

目的通过观察沉默PTEN基因在PC-3细胞中的表达,探讨其对细胞增殖和凋亡的影响。方法设计PTEN si RNA序列,筛选最佳转染效率,Real Time PCR及Western blot检测沉默效率,沉默PTEN基因后,MTT法检测细胞增殖,流式细胞术检测细胞凋亡。结果 Real Time PCR和Western blot结果显示si RNA003沉默效率最高。MTT法显示,si RNA转染组比空白对照组和阴性对照组细胞增殖能力增加(P<0.05),流式细胞术实验显示,si RNA转染组比空白对照组和阴性对照组细胞凋亡降低(P<0.05)。结论 PTEN表达的沉默使PC-3细胞增殖能力上升和凋亡水平下调。

Gouqizi(Fructus Lycii)seed oil reduces D-galactose induced inflammation in testis of rats via Janus kinase 1/signal transducerand activator of transcription 1/nuclear factor-κB in vitro and in vivo

OBJECTIVE:To investigate the efficacy of Gouqizi(Fructus Lycii)seed oil(FLSO)on D-gal induced inflammation in testis of rats in vitro and in vivo.METHODS:In aging Sertoli cells(TM4 cells)induced by D-galactose(D-gal),the expression of upregulated agingrelated proteins.The number of cells counted by cell counting kit(CCK)-8 assay showed a high number of cells disposed with FLSO at 50,100 and 150μg/mL compared to that for the aging model.In vivo,male Sprague–Dawley rats(n=50,8-week-old,230-255 g)were randomly categorized into control,aging model,and FLSO(low-,medium-,and high-dose)groups.The expression of nuclear factor-κB(NF-κB)and its upstream factors[Janus kinase 1(JAK1)and signal transducerand activator of transcription 1(STAT1)]was detected by Western blot and immunofluorescence,related inflammatory factors quantified by enzyme-linked immunosorbent assay.Evaluation of testicular tissue by Johnsen score,the spermatogenic function was explored.RESULTS:The expression of interleukin-1β(IL-1β)(P<0.05),IL-6(P<0.001),and tumor necrosis factorα(TNF-α)(P<0.05)was decreased significantly,while that of heme oxygenase-1(HO-1)(P<0.001)and IL-10(P<0.05)was increased in cells disposed with FLSO 100μg/mL.FLSO inhibited the expression of NF-κB and declined pp65/p65(P<0.01),as detected by Western blotting.In vivo,the levels in serum of IL-1β(P<0.001),IL-6(P<0.05),and TNF-α(P<0.01)declined while IL-10(P<0.05)was upregulated post-FLSO treatment.In addition,the expression of JAK-1 and STAT1 increased significantly in testicular tissue of rats treated with FLSO as compared to the aging model of rats(P<0.001),while the expression of NF-κB(P<0.001)declined in the testis in the FLSO group,as assessed by immunofluorescence.The levels of inhibor B and testosterone in serum both increased(P<0.05).CONCLUSIONS:In conclusion,this study determined the protective effects of FLSO to tolerate inflammatory injury in the testis,indicating that FLSO alleviates inflammation via JAK-1/STAT1/NF-κB pathway.

枸杞籽油对小鼠睾丸支持细胞衰老模型的抗炎作用

目的探讨枸杞籽油对小鼠睾丸支持细胞(TM4)衰老模型的抗炎作用及机制。方法以不同浓度(50、100、150、200 mmol/L)的D-半乳糖(D-Gal)以不同时间(24、36、48 h)体外作用TM4细胞,通过CCK-8法和β-半乳糖苷酶染色筛选最佳时间浓度的D-Gal建立TM4衰老模型,Western blotting检测衰老因子P16^INK4A、P21^Waf1/Cip1、γH2A.X以及半乳糖苷酶蛋白的表达。以不同浓度(1%、3%、5%、10%)枸杞籽油含药血清提前干预TM4细胞衰老模型,并用CCK-8筛选枸杞籽油含药血清最佳浓度。Western blotting检测细胞炎症因子白细胞介素-1(IL-1β)、肿瘤坏死因子(TNF-α)、白细胞介素-6(IL-6)、白细胞介素-10(IL-10)与血红素加氧酶(HO-1)的表达以及p-P65/P65的相对表达量;细胞免疫荧光(IF)定位NF-κB表达。结果D-Gal建立TM4衰老模型结果显示,与正常细胞比较,200 mmol/L浓度的D-Gal干预48 h对细胞增殖有抑制作用,其抑制率为(14.890±0.892)%,差异有统计学意义(F=12.820,P=0.004;t=17.230,P<0.001),同时β-半乳糖苷酶染色结果显示,与正常细胞比较,细胞蓝染阳性率上升(t=5.243,P<0.001)。P16^INK4A(t=2.364,P=0.033)、P21^Waf1/Cip1(t=2.908,P=0.011)、γH2A.X(t=2.511,P=0.025)蛋白表达以及半乳糖苷酶(t=2.299,P=0.037)表达在衰老模型组升高。枸杞籽油抗TM4衰老的结果显示,3%浓度的枸杞籽油含药血清干预衰老TM4细胞后活性上升(t=5.399,P<0.001)。与正常细胞比较,TM4细胞衰老相关蛋白P16^INK4A(t=7.211,P<0.001)、P21^Waf1/Cip1(t=8.248,P<0.001)、γH2A.X(t=4.145,P<0.001)以及半乳糖苷酶(t=7.288,P<0.001)表达下调;与模型组相比,3%浓度的枸杞籽油含药血清干预后炎症因子TNF-α(t=4.992,P<0.001)、IL-1β(t=2.921,P=0.011)、IL-6(t=4.985,P<0.001)的表达下调,HO-1(t=6.047,P<0.001)与IL-10(t=2.315,P=0.036)表达上调;衰老TM4细胞p-P65/P65相对表达量在3%浓度的枸杞籽油含药血清干预后下调(t=24.630,P<0.001)。结论3%枸杞籽油含药血清干预,对衰老TM4细胞有明显的抗炎作用,其机制可能是下调核因子κB进而抑制下游炎症因子IL-1β、TNF-α、IL-6表达,并启动抑炎因子HO-1、IL-10的表达。

Germ cell transplantation in infertility mouse

This work investigated the spermatogenesis in an infertility BALB/c-nu mouse model by reinfusing germline stem cells into seminiferous tubules. Donor germ cells were isolated from male FVB/NJ-GFP transgenic mice. Seminiferous tubule microinjection was applied to achieve intratubular germ cell transfer. The germ cells were injected into exposed testes of the infertility mice. We used green fluo-rescence and DNA analysis of donor cells from GFP transgenic mice as genetic marker. The natural mating and Southern blot methods were applied to analyze the effect of sperm cell transplantation and the sperm function after seminiferous tubule microinjection. The spermatogenesis was morphologi-cally observed from the seminiferous tubules in 41/60 (68.33%) of the injected recipient mice using allogeneic donor cells. In the colonized testes, matured spermatozoa were seen in the lumen of the seminiferous tubules. In this research, BALB/c-nu infertility mouse model, the recipient animal, was used to avoid immunological rejection of donor cells, and germ cell transplantation was applied to overcome infertility caused by busulfan treatment. These results demonstrate that this technique of germ cell transplantation is of great use. Germ cell transplantation could be potentially valuable to oncological patients.