本文選題：半夏瀉心湯 + Cx43-siRNA　； 參考：《北京中醫(yī)藥大學(xué)》2017年碩士論文

【摘要】：[目的]為進(jìn)一步探究半夏瀉心湯對(duì)胃腸運(yùn)動(dòng)障礙的調(diào)節(jié)機(jī)制,以細(xì)胞間縫隙連接通訊為核心,從Cx43與胃腸動(dòng)力障礙的關(guān)系角度,揭示半夏瀉心湯通過(guò)Cx43調(diào)節(jié)胃腸運(yùn)動(dòng)的細(xì)胞分子機(jī)制,進(jìn)而從細(xì)胞間通訊的角度對(duì)"心下痞"的內(nèi)涵進(jìn)行揭示。這不僅可以為半夏瀉心湯的治療作用提供科學(xué)依據(jù),同時(shí)也有助于在機(jī)理探究的基礎(chǔ)上提高半夏瀉心湯調(diào)節(jié)胃腸運(yùn)動(dòng)障礙的療效,促進(jìn)半夏瀉心湯治療消化系統(tǒng)疾病的現(xiàn)代化發(fā)展。[方法]采用SD雄性大鼠灌胃方法,制備半夏瀉心湯含藥血清;同時(shí)去離子水灌胃,制備SD雄性大鼠血清作為對(duì)照。將穩(wěn)定培養(yǎng)傳代后的胃腸間質(zhì)瘤細(xì)胞株(GIST-882),隨機(jī)分為空白對(duì)照組、Cx43-siRNA干擾模型組、半夏瀉心湯含藥血清組(即全方組)。利用CCK-8試劑盒檢測(cè)細(xì)胞活力與細(xì)胞存活率,流式細(xì)胞儀測(cè)定細(xì)胞周期、分析細(xì)胞凋亡,鈣離子熒光探針(Fluo-3 AM)檢測(cè)細(xì)胞內(nèi)鈣離子濃度,免疫印跡法檢測(cè)GIST-882細(xì)胞中縫隙連接蛋白43及其磷酸化MAPK、PKA、PKC、PKG等的蛋白表達(dá)水平,RT-PCR法測(cè)定 GIST-882 細(xì)胞中 Cx43 mRNA、MAPK mRNA、PKAmRNA、PKC mRNA等的表達(dá)。[結(jié)果](1)細(xì)胞活力與細(xì)胞存活率:與空白對(duì)照組相比,模型組的細(xì)胞活力與細(xì)胞存活率顯著下降(P0.05);與模型組對(duì)比,全方組細(xì)胞活力與細(xì)胞存活率有著顯著升高(P0.05)。(2)細(xì)胞凋亡與周期:各組能夠不同程度的促進(jìn)細(xì)胞凋亡,但均無(wú)顯著性差異(P0.05)。各組間細(xì)胞在G1期,模型組上升,全方組下降,但無(wú)顯著性差異(P0.05)。各組細(xì)胞在G2期,與空白對(duì)照組相比,模型組細(xì)胞在此期顯著降低,有統(tǒng)計(jì)學(xué)差異(P0.01);與模型組相比,全方組細(xì)胞在此期周期較高,但無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)。各組細(xì)胞在S期,與空白組相比,模型組在S期降低,但無(wú)統(tǒng)計(jì)學(xué)差異(P0.05);與模型組相比,全方組的周期在此期高于模型組(P0.05),有作用趨勢(shì),但無(wú)統(tǒng)計(jì)學(xué)差異。(3)細(xì)胞內(nèi)鈣離子水平:與空白對(duì)照組對(duì)比,模型組中細(xì)胞內(nèi)鈣離子濃度顯著升高(P0.05);與模型組相比,全方組的細(xì)胞內(nèi)鈣離子濃度顯著下降(P0.05)。(4)相關(guān)蛋白表達(dá)水平:與空白組對(duì)比,模型組Cx43、MAPK的蛋白表達(dá)降低明顯(P0.05),模型組P-Cx43、PKA、PKC、PKG等的蛋白表達(dá)與正常組相比無(wú)顯著性差異(P0.05);與模型組相比,全方組Cx43、MAPK的蛋白表達(dá)明顯高于模型組(P0.01),全方組P-Cx43、PKA、PKC、PKG等的蛋白表達(dá)與模型組相比無(wú)顯著性差異(P0.05)。(5)相關(guān)蛋白mRNA表達(dá):與空白組相比,模型組Cx43mRNA、MAPKmRNA、PKAmRNA的表達(dá)明顯降低,具有統(tǒng)計(jì)學(xué)差異(P0.05);與模型組相比,全方組Cx43 mRNA、PKAmRNA、MAPK mRNA等基因的表達(dá)明顯升高,具有統(tǒng)計(jì)學(xué)差異(P0.05);PKC mRNA的表達(dá)中,與空白組相比,模型組表達(dá)增加;與模型組相比,全方組表達(dá)降低,均沒(méi)有統(tǒng)計(jì)學(xué)差異。[結(jié)論](1)半夏瀉心湯能夠促進(jìn)轉(zhuǎn)染Cx43-siRNA的GIST-882細(xì)胞的增殖,且是通過(guò)細(xì)胞存活周期的增長(zhǎng)而非抑制細(xì)胞凋亡使細(xì)胞存活增長(zhǎng),對(duì)轉(zhuǎn)染Cx43-siRNA的GIST-882細(xì)胞具有一定的調(diào)節(jié)和保護(hù)功能;(2)半夏瀉心湯能夠降低轉(zhuǎn)染Cx43-siRNA的GIST-882細(xì)胞內(nèi)的鈣離子濃度,從而調(diào)節(jié)和保護(hù)第二信使Ca2+通道;(3)半夏瀉心湯能夠增加轉(zhuǎn)染Cx43-siRNA的GIST-882細(xì)胞的Cx43、MAPK蛋白的表達(dá),同時(shí)增強(qiáng)Cx43 mRNA、MAPK mRNA及PKA mRNA等相關(guān)基因的表達(dá),從而調(diào)控細(xì)胞間隙通訊,促進(jìn)胃腸功能障礙恢復(fù)。綜合以上結(jié)論,從現(xiàn)代分子機(jī)制的實(shí)驗(yàn)研究角度說(shuō)明,半夏瀉心湯可以改善細(xì)胞間縫隙連接,增強(qiáng)細(xì)胞間縫隙連接通訊功能,調(diào)節(jié)細(xì)胞內(nèi)第二信使Ca2+通道、MAPK信號(hào)通路及PKA等蛋白激酶,從而促進(jìn)轉(zhuǎn)染Cx43-siRNA后GIST-882細(xì)胞的修復(fù),促進(jìn)胃腸功能障礙的恢復(fù)。這為半夏瀉心湯治療"心下痞"提供了可能性的依據(jù),為該方現(xiàn)代分子生物學(xué)機(jī)制的研究打下了基礎(chǔ)。
[Abstract]:[Objective] to further explore the regulation mechanism of Banxia Xiexin Decoction on gastrointestinal motility disorder, with intercellular gap junction communication as the core, from the angle of the relationship between Cx43 and gastrointestinal motility disorder, the molecular mechanism of regulating gastrointestinal motility by Cx43 is revealed, and then the connotation of "lower heart ruffian" is uncovered from the angle of intercellular communication. It can not only provide scientific basis for the treatment of Banxia Xiexin Decoction, but also help to improve the effect of Banxia Xiexin Decoction on regulating gastrointestinal dyskinesia on the basis of mechanism exploration, promote the modern development of digestive system disease with Banxia Xiexin soup. [Methods] the method of gavage of SD male rats was used to prepare Banxia Xiexin Decoction Serum containing medicine and serum of SD male rats were prepared with deionized water as control. The gastrointestinal stromal tumor cell line (GIST-882) was steadily cultured and divided into blank control group, Cx43-siRNA interference model group, and Banxia Xiexin Decoction serum group (the whole group). The cell viability and cell survival rate were detected by CCK-8 kit. Cell cycle was measured by cytometer, apoptosis was analyzed, calcium ion fluorescence probe (Fluo-3 AM) was used to detect intracellular calcium concentration. The protein expression level of gap connexin 43 and its phosphorylated MAPK, PKA, PKC, PKG in GIST-882 cells was detected by Western blot. Cx43 mRNA in GIST-882 cells was determined by RT-PCR method. MAPK [results] (1) cell viability and cell survival rate: compared with the blank control group, the cell viability and cell survival rate decreased significantly (P0.05). Compared with the model group, the cell viability and cell viability were significantly increased (P0.05). (2) cell apoptosis and cycle: each group could promote apoptosis in different degrees. There was no significant difference (P0.05). The cells in each group were in the G1 phase, the model group rose and the whole group decreased, but there was no significant difference (P0.05). Compared with the blank control group, the cells in each group were significantly lower than those in the blank control group (P0.01). Compared with the model group, the whole group cell cycle was higher in this period, but not statistically significant. Difference (P0.05). Compared with the blank group, the model group decreased in the S phase compared with the blank group, but there was no statistical difference (P0.05). Compared with the model group, the cycle period of the whole group was higher than that of the model group (P0.05), and there was no statistical difference. (3) the intracellular calcium level: compared with the blank control group, the intracellular calcium concentration in the model group was strong. Compared with the model group, the intracellular calcium concentration of the whole group decreased significantly (P0.05). (4) the expression level of related proteins: compared with the blank group, the protein expression of Cx43 and MAPK in the model group decreased significantly (P0.05), and the protein expression of P-Cx43, PKA, PKC and PKG in the model group was not significantly different from that of the normal group (P0.05); and the model group was compared with the model group (P0.05). The protein expression of Cx43 and MAPK in the whole group was significantly higher than that in the model group (P0.01). The protein expression of P-Cx43, PKA, PKC and PKG in the whole group was not significantly different from that of the model group (P0.05). (5) the expression of the related protein mRNA: compared with the blank group, the expression of Cx43mRNA, MAPKmRNA, PKAmRNA was significantly lower than that in the blank group; and the model was statistically different. The expression of Cx43 mRNA, PKAmRNA, MAPK mRNA and other genes in the group was significantly higher than that in the group (P0.05). The expression of the model group increased in the expression of PKC mRNA, compared with the blank group. Compared with the model group, the expression of the whole group decreased, and there was no statistical difference. [Conclusion] (1) Banxia Xiexin soup can promote the GIST-882 transfection of Cx43-siRNA GIST-882. Cell proliferation, and the growth of cell survival cycle, is not inhibited by cell apoptosis to increase cell survival, and has some regulatory and protective functions for Cx43-siRNA transfected GIST-882 cells. (2) Banxia Xiexin soup can reduce the concentration of calcium ions in GIST-882 cells transfected with Cx43-siRNA, thereby regulating and protecting the second messenger Ca2+ (3) (3) Banxia Xiexin Decoction can increase the expression of Cx43 and MAPK protein in transfected GIST-882 cells, and enhance the expression of Cx43 mRNA, MAPK mRNA and PKA mRNA, so as to regulate the intercellular space communication and promote the recovery of gastrointestinal dysfunction. Xiexin Decoction can improve the gap junction of cells, enhance the communication function of gap junction between cells, regulate the second messenger Ca2+ channel, MAPK signaling pathway and PKA protein kinase in cell, thus promote the repair of GIST-882 cells after Cx43-siRNA transfection and promote the restoration of gastrointestinal dysfunction. This provides the Banxia Xiexin Decoction for the treatment of "the lower heart". The basis of this study laid a foundation for the study of modern molecular biology mechanism.

【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R285.5

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 王愛(ài);潘立君;于秀石;李麗;司軍強(qiáng);馬克濤;李新芝;;自發(fā)性高血壓大鼠外周血淋巴細(xì)胞連接蛋白43與炎性因子表達(dá)水平的相關(guān)性研究[J];中國(guó)全科醫(yī)學(xué);2017年02期

2 李慧臻;劉琳;劉華一;趙雙梅;王興章;孔祥茹;杜瀟;;半夏瀉心湯對(duì)PLGC大鼠胃黏膜組織病理學(xué)及HP的影響研究[J];中國(guó)中西醫(yī)結(jié)合消化雜志;2017年01期

3 陳健海;仲婕;王凡;孔桂美;董小耘;朱海航;卜平;;胃腸道Cajal間質(zhì)細(xì)胞起搏功能的研究進(jìn)展[J];中國(guó)病理生理雜志;2017年01期

4 孫學(xué)佳;;半夏瀉心湯治療膽汁反流性胃炎的療效分析[J];中國(guó)繼續(xù)醫(yī)學(xué)教育;2016年22期

5 劉余;龔后武;譚達(dá)全;尹抗抗;;半夏瀉心湯加減治療消化性潰瘍的Meta分析[J];中華中醫(yī)藥雜志;2016年08期

6 鄭敏;唐艷萍;;半夏瀉心湯為主治療反流性食管炎的Meta分析[J];世界中西醫(yī)結(jié)合雜志;2016年05期

7 徐云瑩;楊亦奇;;半夏瀉心湯治療消化道腫瘤的機(jī)理淺析[J];云南中醫(yī)中藥雜志;2016年04期

8 李思迪;侯信;亓洪昭;趙瑾;原續(xù)波;;外泌體:為高效藥物投遞策略提供天然的內(nèi)源性納米載體[J];化學(xué)進(jìn)展;2016年Z2期

9 萬(wàn)淼;金哲;;蛋白激酶G對(duì)心肌再灌注損傷及左室重構(gòu)的影響[J];中國(guó)繼續(xù)醫(yī)學(xué)教育;2016年03期

10 曾耀明;余維微;葉海瀟;鄭偉偉;陳志堅(jiān);林上助;;半夏瀉心湯對(duì)寒熱錯(cuò)雜型慢性淺表性胃炎患者胃黏膜肥大細(xì)胞的影響[J];浙江中醫(yī)雜志;2016年01期

，

本文編號(hào)：1888918