细胞外 pH 值及其波动对高磷诱导的大鼠血管平滑肌细胞凋亡的作用及机制研究
세포외 pH 치급기파동대고린유도적대서혈관평활기세포조망적작용급궤제연구
Effects of Extracellular pH and Its Fluctuation on High - phosphate - induced Apoptosis of Rat Vascular Smooth Muscle Cells and Its Mechanism
  • 万方数据
    中国全科医学 중국전과의학
    Chinese General Practice
    2015年 33期 4090-4095 ,共6页

    崔立文%徐金升%白亚玲%张俊霞%张胜雷

    최립문%서금승%백아령%장준하%장성뢰

    肌细胞,平滑肌%细胞凋亡%血管钙化%酸碱环境%半胱氨酸天冬氨酸蛋白酶3 肌細胞,平滑肌%細胞凋亡%血管鈣化%痠堿環境%半胱氨痠天鼕氨痠蛋白酶3
    기세포,평활기%세포조망%혈관개화%산감배경%반광안산천동안산단백매3
    Myocytes,smooth muscle%Apoptosis%Vascular calcification%Acid - base environment%Caspase 3
    目的：探讨细胞外 pH 值的变化及其波动对高磷诱导的大鼠血管平滑肌细胞（VSMCs）凋亡的作用及机制。方法2014年11月—2015年3月，选取8～10周龄健康雄性 SD 大鼠6只。采用简单随机抽样法选取2只大鼠，体外分离培养大鼠胸主动脉 VSMCs。采用随机数字表法将 VSMCs 分为 pH 7.4组和高磷组，高磷组又分为pH 6.8＋磷组、pH 7.4＋磷组、pH 7.7＋磷组和 pH 6.8/7.7＋磷组。pH 7.4组采用含10％胎牛血清（FBS）的 DMEM培养基培养。高磷组采用高磷培养基（10 mmol/ L β-甘油磷酸）培养，并根据数字酸度计的读取值酌情加入1 mol/ L HCl 或7.4％ NaHCO3调整 pH 值；pH 6.8/7.7＋磷组第1天给予 pH 7.7刺激、第2天给予 pH 6.8刺激。将剩余4只大鼠按照上述方法培养，重复2次。采用免疫组织化学（SP 法）进行细胞鉴定。采用反转录聚合酶链反应（RT - PCR）法检测半胱氨酸的天冬氨酸蛋白水解酶3（ Caspase -3） mRNA 相对表达水平及 B 淋巴细胞瘤2（ Bcl -2） mRNA/ Bcl -2相关 X 蛋白（Bax）mRNA。采流式细胞仪检测细胞凋亡率。结果倒置相差显微镜下可见：大鼠胸主动脉组织块贴壁3～4 d 后细胞从组织块边缘爬出，细胞为长梭形，呈束状排列；5～6 d 成同心圆状细胞团，形成明显的细胞生长晕，并围绕组织块边缘呈束状、放射状延伸；6～7 d 后细胞融合成片，细胞体梭形或不规则三角形，细胞质向外伸出2～3个长短不等的突起。免疫组织化学检测可见：细胞质表达强阳性。pH 6.8＋磷组Caspase -3 mRNA 表达水平低于pH 7.4组，pH 7.4＋磷组、pH 7.7＋磷组Caspase -3 mRNA 表达水平高于 pH 7.4组（P ＜0.05）；pH 7.4＋磷组、pH 7.7＋磷组、pH 6.8/7.7＋磷组 Caspase -3 mRNA 表达水平高于 pH 6.8＋磷组（ P ＜0.05）；pH 7.7＋磷组Caspase -3 mRNA 表达水平高于 pH 7.4＋磷组，pH 6.8/7.7＋磷组 Caspase -3 mRNA 表达水平低于pH 7.4＋磷组、pH 7.7＋磷组（P ＜0.05）。pH 7.4＋磷组、pH 7.7＋磷组、pH 6.8/7.7＋磷组 Bcl -2 mRNA/ Bax mRNA 分别小于 pH 7.4组、pH 6.8＋磷组（P ＜0.05）；pH 7.7＋磷组Bcl -2 mRNA/ Bax mRNA 小于pH 7.4＋磷组（ P ＜0.05）；pH 6.8/7.7＋磷组 Bcl -2 mRNA/ Bax mRNA 大于pH 7.7＋磷组。pH 6.8＋磷组、pH 7.4＋磷组、pH 7.7＋磷组、pH 6.8/7.7＋磷组细胞凋亡率大于 pH 7.4组（ P ＜0.05）；pH 7.4＋磷组、pH 7.7＋磷组、pH 6.8/7.7＋磷组细胞凋亡率大于pH 6.8＋磷组（P ＜0.05）；pH 7.7＋磷组细胞凋亡率大于 pH 7.4＋磷组，pH 6.8/7.7＋磷组细胞 凋 亡 率 小 于pH 7.4＋磷组（P ＜0.05）；pH 6.8/7.7＋磷组细胞凋亡率小于pH 7.7＋磷组（P ＜0.05）。结论细胞外酸性环境能够抑制高磷培养的大鼠 VSMCs 凋亡，而细胞外碱性环境能够促进其凋亡。其具体机制可能是影响了 Bcl -2、Bax 的表达水平进而影响了 Caspase -3的表达水平，从而影响了细胞凋亡。
    목적：탐토세포외 pH 치적변화급기파동대고린유도적대서혈관평활기세포（VSMCs）조망적작용급궤제。방법2014년11월—2015년3월，선취8～10주령건강웅성 SD 대서6지。채용간단수궤추양법선취2지대서，체외분리배양대서흉주동맥 VSMCs。채용수궤수자표법장 VSMCs 분위 pH 7.4조화고린조，고린조우분위pH 6.8＋린조、pH 7.4＋린조、pH 7.7＋린조화 pH 6.8/7.7＋린조。pH 7.4조채용함10％태우혈청（FBS）적 DMEM배양기배양。고린조채용고린배양기（10 mmol/ L β-감유린산）배양，병근거수자산도계적독취치작정가입1 mol/ L HCl 혹7.4％ NaHCO3조정 pH 치；pH 6.8/7.7＋린조제1천급여 pH 7.7자격、제2천급여 pH 6.8자격。장잉여4지대서안조상술방법배양，중복2차。채용면역조직화학（SP 법）진행세포감정。채용반전록취합매련반응（RT - PCR）법검측반광안산적천동안산단백수해매3（ Caspase -3） mRNA 상대표체수평급 B 림파세포류2（ Bcl -2） mRNA/ Bcl -2상관 X 단백（Bax）mRNA。채류식세포의검측세포조망솔。결과도치상차현미경하가견：대서흉주동맥조직괴첩벽3～4 d 후세포종조직괴변연파출，세포위장사형，정속상배렬；5～6 d 성동심원상세포단，형성명현적세포생장훈，병위요조직괴변연정속상、방사상연신；6～7 d 후세포융합성편，세포체사형혹불규칙삼각형，세포질향외신출2～3개장단불등적돌기。면역조직화학검측가견：세포질표체강양성。pH 6.8＋린조Caspase -3 mRNA 표체수평저우pH 7.4조，pH 7.4＋린조、pH 7.7＋린조Caspase -3 mRNA 표체수평고우 pH 7.4조（P ＜0.05）；pH 7.4＋린조、pH 7.7＋린조、pH 6.8/7.7＋린조 Caspase -3 mRNA 표체수평고우 pH 6.8＋린조（ P ＜0.05）；pH 7.7＋린조Caspase -3 mRNA 표체수평고우 pH 7.4＋린조，pH 6.8/7.7＋린조 Caspase -3 mRNA 표체수평저우pH 7.4＋린조、pH 7.7＋린조（P ＜0.05）。pH 7.4＋린조、pH 7.7＋린조、pH 6.8/7.7＋린조 Bcl -2 mRNA/ Bax mRNA 분별소우 pH 7.4조、pH 6.8＋린조（P ＜0.05）；pH 7.7＋린조Bcl -2 mRNA/ Bax mRNA 소우pH 7.4＋린조（ P ＜0.05）；pH 6.8/7.7＋린조 Bcl -2 mRNA/ Bax mRNA 대우pH 7.7＋린조。pH 6.8＋린조、pH 7.4＋린조、pH 7.7＋린조、pH 6.8/7.7＋린조세포조망솔대우 pH 7.4조（ P ＜0.05）；pH 7.4＋린조、pH 7.7＋린조、pH 6.8/7.7＋린조세포조망솔대우pH 6.8＋린조（P ＜0.05）；pH 7.7＋린조세포조망솔대우 pH 7.4＋린조，pH 6.8/7.7＋린조세포 조 망 솔 소 우pH 7.4＋린조（P ＜0.05）；pH 6.8/7.7＋린조세포조망솔소우pH 7.7＋린조（P ＜0.05）。결론세포외산성배경능구억제고린배양적대서 VSMCs 조망，이세포외감성배경능구촉진기조망。기구체궤제가능시영향료 Bcl -2、Bax 적표체수평진이영향료 Caspase -3적표체수평，종이영향료세포조망。
    Objective To investigate the effect of extracellular pH changes and its fluctuations on high - phosphate -induced apoptosis of rat vascular smooth muscle cells(VSMCs)and its mechanism. Methods A total of 6 healthy male SD rats aged 8 - 10 weeks were selected from November 2014 to March 2015. Using simple random sampling method,we selected 2 rats,&nbsp;and VSMCs from rat thoracic aorta were cultured in vitro. Using random number table method,VSMCs were divided into pH 7. 4 group and high - phosphate group. High - phosphate group was divided into pH 6. 8 + phosphate group,pH 7. 4 + phosphate group,pH 7. 7 + phosphate group and pH 6. 8 / 7. 7 + phosphate group. DMEM culture medium containing 10% FBS was employed in pH 7. 4 group. High - phosphate culture medium was employed in high - phosphate group,and according to the read- out values of acidity meter,1 mol/ L HCl or 7. 4% NaHCO3 were added to adjust pH;pH 6. 8 / 7. 7 + phosphate group was stimulated with pH 7. 7 on day 1 and was given pH 6. 8 on day 2. The VSMCs of the rest 4 rats were cultured with the above method for two times. Cells were determined using immunohistochemistry SP method,RT - PCR was employed to detect the mRNA relative expression level of Caspase - 3 and Bcl - 2 mRNA/ Bax mRNA. Flow cytometry was employed to detect apoptosis rate. Results Under inverted phase contrast microscope,the observation results were as follows:3 - 4 d later,VSMCs crawled out of the rim of tissue block in long fusiform shape and sarciniform distribution;5 - 6 d later,cell mass was formed in the shape of concentric circle with obvious growing follicle,crawling around the rim of tissue block in sarciniform and radiated shape;6 -7 d later,the cells were merged into films,and the cell body took on fusiform shape or irregular triangular shape with cytoplasm stretching out forming 2 to 3 protrusions of different lengths. The pH 6. 8 + phosphate group was lower than pH 7. 4 group in Caspase - 3 mRNA expression(P < 0. 05),and pH 7. 4 + phosphate group and pH 7. 7 + phosphate group were higher than pH 7. 4 group in Caspase - 3 mRNA expression(P < 0. 05);pH 7. 4 + phosphate group,pH 7. 7 + phosphate group and pH 6. 8/ 7. 7 + phosphate group were higher than pH 6. 8 + phosphate group in Caspase - 3 mRNA expression( P < 0. 05);pH 7. 7+ phosphate group was higher than pH 7. 4 + phosphate group in Caspase - 3 mRNA expression(P < 0. 05),and pH 6. 8 / 7. 7+ phosphate group was lower than pH 7. 4 + phosphate group in Caspase - 3 mRNA expression( P < 0. 05);pH 6. 8 / 7. 7+ phosphate group was lower than pH 7. 7 + phosphate group in Caspase - 3 mRNA expression ( P < 0. 05 ) . pH 7. 4+ phosphate group,pH 7. 7 + phosphate group and pH 6. 8 / 7. 7 + phosphate group were lower than pH 7. 4 + phosphate group and pH 6. 8 + phosphate group in Bcl - 2 mRNA/ Bax mRNA(P < 0. 05);pH 7. 7 + phosphate group was lower than pH 7. 4+ phosphate group in Bcl - 2 mRNA/ Bax mRNA( P < 0. 05);pH 6. 8 / 7. 7 + phosphate group was higher than pH 7. 7+ phosphate group in Bcl - 2 mRNA/ Bax mRNA. pH 6. 8 + phosphate group,pH 7. 4 + phosphate group,pH 7. 7 + phosphate group and pH 6. 8 / 7. 7 + phosphate group were higher than pH 7. 4 group in cell apoptosis rate ( P < 0. 05 );pH 7. 4+ phosphate group,pH 7. 7 + phosphate group and pH 6. 8 / 7. 7 + phosphate group were higher than pH 6. 8 group in cell apoptosis rate(P < 0. 05);pH 7. 7 + phosphate group was higher than pH 7. 4 + phosphate group in cell apoptosis rate(P <0. 05),and pH 6. 8 / 7. 7 + phosphate group was lower pH 7. 4 + phosphate group in cell apoptosis rate(P < 0. 05);pH 6. 8/ 7. 7 + phosphate group was lower than pH 7. 7 + phosphate group in cell apoptosis rate(P < 0. 05). Conclusion Extracellular acidic environment can inhibit high - phosphorus - induced VSMCs apoptosis,whereas extracellular alkaline environment and pH fluctuations induce apoptosis. The mechanism may be that pH value may influence Bcl - 2 and Bax expression and further influence Caspase - 3 expression,thus influencing cell apoptosis.
    원문보기 원문다운로드 사이트로이동
저자의 최근 논문