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范 敏,郑文兰,文晓敏.加味宫外孕 II 号方对人滋养叶细胞 Caspase-12 通路影响的研究[J].中国比较医学杂志,2020,30(8):29~34.
加味宫外孕 II 号方对人滋养叶细胞 Caspase-12 通路影响的研究
Effect of modified ectopic pregnancy formula II on the Caspase-12 pathway in human trophoblasts
投稿时间:2020-01-06  
DOI:10. 3969 / j.issn.1671-7856. 2020. 08. 005
中文关键词:  加味宫外孕 II 号方  内质网应激  HTR-8/ SVneo 细胞  半胱氨酸天冬氨酸蛋白酶-7  半胱氨酸天冬 氨酸蛋白酶-9  半胱氨酸天冬氨酸蛋白酶-3
英文关键词:modified ectopic pregnancy formula II  endoplasmic reticulum stress  HTR-8/ SVneo cells  Caspase-7  Caspase-9  Caspase-3
基金项目:
作者单位E-mail
范 敏 贵州中医药大学,贵阳 550025 fanmin1207@ 163.com 
郑文兰 贵州中医药大学第一附属医院,贵阳 550001 yunqq533@ 163.com 
文晓敏 贵州中医药大学第一附属医院,贵阳 550001  
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中文摘要:
      目的 观察加味宫外孕 II 号方对人滋养叶细胞内质网应激 Caspase-12 信号通路凋亡相关蛋白 Caspase-7、Caspase-9、Caspase-3 水平的影响及细胞超微结构变化,探讨其治疗异位妊娠的作用机制。 方法 将 SD 大鼠分为阴性对照组(B 组)、低、中、高剂量中药组(C 组、D 组、E 组)、甲氨蝶呤组(F 组)、中西药结合组(G 组), 予相应药物处理后取血制备含药血清,将各组含药血清分别作用于 HTR-8/ SVneo 细胞 24 h,并设置未用含药血清处理的细胞作为空白对照组(A 组)。采用蛋白质免疫印迹法(Western blot)与实时荧光定量 PCR(Real-time PCR) 检测 HTR-8/ SVneo 细胞中凋亡基因 Caspase-7、Caspase-9、Caspase-3 蛋白及 mRNA 的表达水平;采用透射电镜观察 细胞超微结构的变化。 结果 (1)Caspase-7、Caspase-9、Caspase-3 蛋白水平及 mRNA 相对表达量:A 组与 B 组比较 无差异(P>0. 05);与 B 组比较,C 组 Caspase-7 mRNA、Caspase-9 蛋白及 Caspase-3 蛋白与 mRNA 表达均上调明显 (P<0. 05),而 Caspase-7 蛋白、Caspase-9 mRNA 表达无差异(P>0. 05);D、E、F、G 组 Caspase-7、Caspase-9、Caspase-3 蛋白及 mRNA 表达量均较 B 组明显上调(P<0. 05);E 组较 D 组上调明显(P<0. 05);E 组与 F 组比较,Caspase-7、 Caspase-9 蛋白与 mRNA 及 Caspase-3 蛋白表达上调明显(P<0. 05);E 组与 G 组比较,Caspase-9 蛋白与 mRNA 及 Caspase-3 蛋白表达上调明显(P<0. 05);F 组与 G 组比较无差异(P>0. 05)。 (2)透射电镜结果显示:A 组与 B 组细 胞染色质均匀散布于核膜内,细胞内质网、线粒体、高尔基体清晰可见;与 B 组比较,C、D、E、F、G 组细胞微绒毛减 少,染色质固缩,异染色质边缘化,部分线粒体肿胀、脊紊乱或断裂、甚至消失,线粒体空泡化等典型的凋亡特征改 变。 结论 加味宫外孕 II 号方可能通过激活内质网应激 Caspase-12 信号通路,破坏滋养叶细胞的形态学结构,上 调 Caspase-7、Caspase-9、Caspase-3 凋亡相关基因的表达,促进滋养叶细胞凋亡。
英文摘要:
      Objective To examine the effect of modified ectopic pregnancy formula II on levels of apoptosis- related proteins Caspase-7, Caspase-9, and Caspase-3 in the endoplasmic reticulum stress Caspase-12 signaling pathway in human trophoblasts, as well as associated changes in cell ultrastructure, to explore its treatment mechanism for ectopic pregnancy. Methods SD rats were divided into a negative control group ( group B), low-, medium-, and high-dose Chinese medicine groups ( groups C, D, E), a methotrexate group ( group F), and a combined Chinese and Western medicine group ( group G). After receiving the corresponding drug treatment, we collected blood to prepare a drug- containing serum. The drug-containing serum from each group was applied to HTR-8/ SVneo for 24 h, and cells not treated with drug-containing serum were set as a blank control group (group A). The expression levels of apoptotic genes Caspase- 7, Caspase-9, Caspase-3 protein and mRNA in HTR-8/ SVneo cells were detected via Western blot and real-time quantitative PCR. Transmission electron microscopy was used to observe changes in cell ultrastructure. Results (1) There was no difference between group A and group B in terms of Caspase-7, Caspase-9, or Caspase-3 protein levels and relative mRNA expression (P> 0. 05). Compared with group B, expression levels of Caspase-7 mRNA, Caspase-9 protein, and Caspase-3 protein and mRNA were significantly up-regulated in group C ( P < 0. 05), but we found no differences in Caspase-7 protein or Caspase-9 mRNA expression (P> 0. 05). In terms of Caspase-7, Caspase-9, and Caspase-3 protein and mRNA expression, expression was significantly increased in groups D, E, F, and G compared with group B (P < 0. 05), and expression in Group E was significantly up-regulated compared with group D ( P < 0. 05). Group E was compared with group F, expression levels of Caspase-7, Caspase-9 protein and mRNA, and Caspase-3 protein were significantly up-regulated (P < 0. 05). Group E was compared with group G, expression of Caspase-9 protein and mRNA and Caspase-3 protein was significantly increased (P <0. 05). There was no difference between group F and group G (P> 0. 05). ( 2) Transmission electron microscopy indicated that the cell chromatin in group A and group B was evenly distributed in the nuclear membrane, and the endoplasmic reticulum, mitochondria, and Golgi apparatus were clearly visible. Compared with group B, the cell microvilli in groups C, D, E, F, and G were reduced, chromatin was condensed, heterochromatin was marginalized, some mitochondria were swollen, disordered, broken, or even absent, and mitochondrial vacuolation were typical apoptotic characteristics. Conclusions Modified ectopic pregnancy formula II may activate the Caspase-12 signaling pathway of endoplasmic reticulum stress, destroy the morphological structure of trophoblasts, up-regulate the expression of Caspase-7, Caspase-9, and Caspase-3 apoptosis-related genes, and promote trophoblast apoptosis.
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