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橘荔散结丸有效部位对子宫肌瘤细胞增殖、凋亡及相关因子表达的影响
The Research of the Effects of Juli Sanjie Pill on Proliferation, Apoptosis, Related Factors Expression Levels in Uterine Leiomyoma Cells
【作者】 李坤寅；
【导师】 尤昭玲；
【作者基本信息】 湖南中医药大学 ， 中医妇科学， 2010， 博士
【摘要】 目的：探讨橘荔散结丸各提取部位对子宫肌瘤细胞增殖、凋亡及PR-A、EGF、EGFR表达水平的影响,明确其具体有效部位,为药物筛选、精简处方奠定基础。方法：1.采用胶原酶消化法对人子宫肌瘤细胞进行原代培养。2.采用现代技术提取橘荔散结丸的化学成分群。3.运用MTT法、流式细胞术、免疫细胞化学、免疫细胞荧光、酶联免疫等技术检测橘荔散结丸干预后子宫肌瘤细胞的增殖、凋亡、PR-A、EGF及EGFR的表达变化。结果：1.成功培养了人子宫肌瘤细胞。2.与空白对照组相比：1)橘荔散结丸醇提物、水提醇沉液、水提醇沉物及挥发油组OD值均显著降低,差异有统计学意义(p<0.05),呈浓度依赖关系(p<0.05)。2)橘荔散结丸各提取部位组的凋亡率呈上升趋势,其中醇提物组的凋亡率上升显著,有统计学意义(p<0.05)；其他各提取部位组凋亡率的差异无统计学意义(p>0.05)。3)各橘荔散结丸提取部位组的PR-A阳性表达细胞的MOD呈显著降低,差异有统计学意义(p<0.05)。4)高浓度醇提物、水提醇沉液及挥发油组EGF分泌均显著下降,差异有统计学意义(p<0.05)；水提醇沉物组EGF分泌差异无统计学意义(p>0.05)。5)橘荔散结丸醇提物、水提醇沉液、水提醇沉物及挥发油组EGFR阳性表达细胞的MOD均显著降低,差异有统计学意义(p<0.05),挥发油组间成浓度依赖关系(p<0.05)。3.与阳性对照RU486组相比：橘荔散结丸的醇提物、水提醇沉液、水提醇沉物及挥发油的高浓度组的EGFR表达显著降低,差异有统计学意义(p<0.05)。结论：本研究表明降低子宫肌瘤细胞的增殖能力,诱导子宫肌瘤细胞凋亡而使其自然缩小或消退,降低子宫肌瘤细胞PR-A、EGFR的表达及EGF的分泌,可能是橘荔散结丸治疗子宫肌瘤的主要机理之一。而在橘荔散结丸的各组提取部位中,醇提物组的贡献较大。
【Abstract】 (Hunan College of Traditional Chinese Medicine, Changsha,410007)Objects:To explore the influence of Juli Sanjie Pill (JSP)on proliferation, apoptosis, progesterone receptor A (PR-A), EGF, EGFR expression level of Uterine Leiomyoma Cells (ULCs), observe the Effective parts, and provide a more scientific basis on Drug screening and streamlining the prescription.Method:1. Uterine Leiomyoma Cells (ULCs) were digested with collagenase in primary culture.2. The chemical composition of groups JSP was extracted by modern technology.3. MTT, Flow cytometry, immunocytochemistry, immunofluorescence and euzymelinked immunosorbent assay (ELISA) were performed to observe the expression and changes on proliferation, apoptosis, PR-A, EGF, EGFR of ULCs, which were interved by JSP.Result:1. ULCs were successfully cultured.2.1) Compared with the blank group, OD of the alcohol extraction group. the water extraction and alcohol precipitation liquid group、the sediments from the water and alcohol group and the volatile oil group of JSP were significantly lower and their difference which was a dose-dependent was statistically significant (p<0.05).2) There was significant difference between the blank group and the alcohol extraction group in the rate of apoptosis (p<0.05), and there was no statistical significance in other extracts group of JPS (p>0.05). However, compared with the blank group, the rate of apoptosis in the four group of JPS grown more quickly.3) The difference was significant difference between the blank group and the four group of JPS in the MOD expression of ULCs where PR-A was positive (p<0.05).4) Compared with the blank group, the secretion of EGF in the high concentrations of the extraction group、the water extraction and alcohol precipitation liquid group and the volatile oil group of JSP were significantly decreased and the difference was statistically significant (p<0.05), but there was no significant difference between the blank group and the sediments from the water and alcohol group (p>0.05).5) The four group of JPS were significantly lower EGFR expression comparing with the blank group (p<0.05), and the EGFR expression of the two volatile oil group was significantly different (p<0.05).3. Compared with the RU486 group, EGFR of the alcohol extraction group、the water extraction and alcohol precipitation liquid group、the sediments from the water and alcohol group and the volatile oil group of JSP were significantly lower (p<0.05).Conclusion:It indicates that it is one of mechanisms for Juli Sanjie Pill (JSP) to treat Uterine Leiomyoma to induce apoptosis of the ULCs, make it spontaneous regression, make PR-A、EGFR and EGF fall in ULCs. The role of the extraction group was more significant.