顺铂通过诱导膀胱癌细胞自噬促进细胞凋亡

目的：研究自噬在顺铂引起膀胱癌细胞凋亡中的作用。方法：以膀胱癌细胞T24为细胞模型,利用透射电镜观察自噬泡形成,荧光显微镜观察转染绿色荧光蛋白和微管相关蛋白1轻链3融合蛋白（green fluorescent protein and microtubule associated protein 1 light chain 3 fusion protein,GFP-LC3）的质粒的荧光聚集情况。应用蛋白质免疫印迹检测LC3-Ⅱ的积累,检测顺铂能否诱导膀胱癌T24细胞发生自噬。此外,蛋白质免疫印迹还被用来检测自噬相关信号通路哺乳动物雷帕霉素受体（mammal target of rapamycin, mTOR）及其下游相对分子质量70 000的核糖体蛋白质S6激酶（70 000 ribosomal protein S6 kinase,P70S6K）的变化,以及凋亡标志蛋白聚腺苷二磷酸核糖聚合酶(poly ADP-ribose polymerase,PARP)的切割,之后利用3-(4,5-二甲基)-5-(3-羧甲基苯环)-2-(4-硫基苯)-2H-四唑盐复合物检测法［ 3-( 4, 5-dimethylthiazol-2-yl)-5-( 3-carboxymethoxyphenyl)-2-( 4-sulfophenyl)-2H- tetrazolium, inner salt,MTS］观察在自噬促进剂雷帕霉素（rapamycin）存在与否的情况下顺铂引起的膀胱癌细胞活力变化。本实验还使用了RNA干扰技术敲降LC3表达。结果：电子显微镜显示相比对照组,顺铂可以引起膀胱癌细胞出现大量自噬泡。荧光显微镜观察GFP-LC3聚集程度,顺铂组也明显高于对照组。蛋白质免疫印迹检测LC3的结果发现顺铂组的LC3-Ⅱ含量随时间延长和顺铂浓度增加而明显增加,尤其是在48 h时,50与100 μmol/L顺铂处理下LC3-Ⅱ/Actin（%）的灰度值分别升高了30和44,而mTOR/P70S6K的磷酸化也受到顺铂处理的抑制,在48 h,100 μmol/L顺铂处理下其磷酸化条带几乎完全被抑制。MTS结果发现顺铂可导致细胞活力的丢失,在50和100 μmol/L顺铂处理24 h时分别下降了12%和35%,而且用自噬促进剂雷帕霉素和顺铂联合处理组的细胞活力丢失大于单用顺铂处理的对照组（F=74.890,P<0.01）。RNA干扰实验显示,敲降自噬相关基因LC3,可减少顺铂引起的PARP剪切,细胞凋亡减少。结论：发现顺铂可以引起膀胱癌细胞T24发生自噬,并发现该自噬的作用能促进顺铂诱导的细胞凋亡。     

研究四肢创伤骨折后骨不连应用植入物内固定治疗的效果

目的:探析不同植入物内固定治疗四肢创伤骨折后骨不连的效果。方法:选取2016年1月～2017年10月我院骨科收治的78例四肢创伤骨折后骨不连患者为研究对象,采用随机分配原则分组,各39例。对照组用动力加压钢板内固定治疗,观察组用带锁髓内钉固定治疗,对两组的治疗效果进行比较。结果:观察组的治疗优良率为94.87%,明显高于对照组的76.92%,两组差异显著(P0.05);观察组的骨折愈合时间明显短于对照组,两组有显著差异(P0.05)。结论:带锁髓内钉固定治疗四肢创伤骨折后骨不连的效果更为显著,且可缩短骨折的愈合时间,预后佳,值得进一步推广。     

家族性腺瘤性息肉病5例诊疗分析

家族性腺瘤性息肉病(FAP)是少见病,表现为大肠内成百上千的腺瘤性息肉,患者及其家族是发生大肠癌的高发人群,约占大肠癌的1%。因此,对患者家系进行筛选是早期诊[1]     

中低位直肠癌保肛手术临床分析

目的探讨保肛术治疗低位直肠癌患者的临床效果。方法回顾性分析2002年5月至2009年5月期间中低位直肠癌保肛手术治疗的58例患者的临床资料,所有患者均经过手术病理确诊。手术遵循直肠癌根治切除术和直肠系膜全切除术的原则,远近切缘距肛瘤分别至少达2.5cm及8cm,应用双吻合器法于骶前行结直肠端端吻合。结果 58例全部完成保肛手术,2例术后2周内发生吻合口漏,3例发生吻合口狭窄,无手术死亡。平均随访3年,局部复发3例。结论严格掌握保肛手术指征、合理选择患者、规范手术是中低位直肠癌保肛根治手术成功的关键。     

小切口治疗皮下血肿16例体会

我院自1997年至今收入臀部、小腿、腰背部巨大血肿16例，现报道如下。     

不液化精液体外处理的初步研究

①目的探讨体外处理不液化精液的方法。②方法随机选取不液化精液标本１５份，分别应用α－淀粉酶、淋巴细胞分离液（Ｆｉｃｏｌ液）及牛血清白蛋白（ＢＳＡ）处理标本并设置对照组，观察精子活动率、前向运动精子百分数和精子穿透力的变化。③结果经Ｆｉｃｏｌ液及ＢＳＡ处理后的精液精子活动率、前向运动精子百分数和精子穿透力较未处理组有显著提高（ｔ＝６．０９～２９．６６，Ｐ均＜０．０１）；经α－淀粉酶处理后的精液，前向运动精子百分数和精子穿透力较未处理组明显增高（ｔ＝６．０２～９．６７，Ｐ均＜０．０１）。④结论用Ｆｉｃｏｌ液及ＢＳＡ处理不液化精液，可以得到高质量的精子，有望在临床治疗精液不液化不育症方面推广应用     

上腹部疼痛2月余--思考病例(120)讨论部分

1病历摘要详见本刊2005年第1期第46页。2临床讨论本例有如下特点:①中年男性;②既往体健,无肝炎、结核等病史;③持续上腹部疼痛2月余而入院,经外院及入院后应用抗生素及抑酸治疗等无效;体格检查腹部脐上稍偏右深压痛,无反跳痛;⑤外院腹部CT示胰头可疑饱满,彩色多普勒超声(彩超)     

雄激素剥夺条件下阻断自噬增加LNCaP细胞凋亡

Objective To investigate the change of apoptosis in LNCaP cells after inhibition of autophagic process under androgen removal conditions. Methods The autophagic level was deter-mined by using confocal microscopy and RT-PCR. The DAPI staining was used to indicate the apopto-sis of LNCaP cells after inhibition of autophagic by 3-MA. Also, Z-VAD-FMK was used to extend the apoptosis results. Results ①Androgen deprivation led to increased autophagy in LNCaP cells. LN-CaP cells cultured in complete medium(CM) presented low autophagic process with 1.9 scores. After 24 hours, the punetate GFP-LC3 structures were accumulated in the cells cultured in serum-free medi-um (SF)(2.64 scores). In contrast, the number of punctate GFP-LC3 remained at a very low level (1.85 scores), when cells were incubated with DHT in SFA(serum-free medium+DHT). Statistical analysis showed the significant difference between SF and SFA (P<0.01). Semiquantitative RT-PCR was employed to examine the mRNA expression of LC3. Indeed, cells grown in the medium without serum had a higher LC3 mRNA expression with the highest at 12 hour time point as compared with the cells grown in CM. DHT treatment reduced the level of LC3 mRNA. ②Blockage of autophagy by 3-MA increased the apoptosis of LNCaP cells. LNCaP cells in SF and SFA just presented a basal level of apoptosis, which is (3.19±1.09)% and (3.01±0.33)% , respectively. Under androgen-free con-ditions, inhibition of autophagy by 3-MA could increase apoptosis significantly(10. 90±2.91%). While Z-VAD-FMK, a pan Caspase inhibitor, was able to suppress this apoptotic process to the level of (1.16±0.52)%, which was statistically significant(P<0.01). Conclusions Androgen removal can lead to the increase of autophagy in LNCaP cells. Moreover, inhibition of autophagy promotes the occurrence of apoptosis.     

雄激素剥夺条件下阻断自噬增加LNCaP细胞凋亡

Objective To investigate the change of apoptosis in LNCaP cells after inhibition of autophagic process under androgen removal conditions. Methods The autophagic level was deter-mined by using confocal microscopy and RT-PCR. The DAPI staining was used to indicate the apopto-sis of LNCaP cells after inhibition of autophagic by 3-MA. Also, Z-VAD-FMK was used to extend the apoptosis results. Results ①Androgen deprivation led to increased autophagy in LNCaP cells. LN-CaP cells cultured in complete medium(CM) presented low autophagic process with 1.9 scores. After 24 hours, the punetate GFP-LC3 structures were accumulated in the cells cultured in serum-free medi-um (SF)(2.64 scores). In contrast, the number of punctate GFP-LC3 remained at a very low level (1.85 scores), when cells were incubated with DHT in SFA(serum-free medium+DHT). Statistical analysis showed the significant difference between SF and SFA (P<0.01). Semiquantitative RT-PCR was employed to examine the mRNA expression of LC3. Indeed, cells grown in the medium without serum had a higher LC3 mRNA expression with the highest at 12 hour time point as compared with the cells grown in CM. DHT treatment reduced the level of LC3 mRNA. ②Blockage of autophagy by 3-MA increased the apoptosis of LNCaP cells. LNCaP cells in SF and SFA just presented a basal level of apoptosis, which is (3.19±1.09)% and (3.01±0.33)% , respectively. Under androgen-free con-ditions, inhibition of autophagy by 3-MA could increase apoptosis significantly(10. 90±2.91%). While Z-VAD-FMK, a pan Caspase inhibitor, was able to suppress this apoptotic process to the level of (1.16±0.52)%, which was statistically significant(P<0.01). Conclusions Androgen removal can lead to the increase of autophagy in LNCaP cells. Moreover, inhibition of autophagy promotes the occurrence of apoptosis.