中国全科医学
中國全科醫學
중국전과의학
Chinese General Practice
2015年
27期
3294-3299
,共6页
李同妙%徐金升%冯雨%张胜雷%张俊霞%崔立文%张慧然
李同妙%徐金升%馮雨%張勝雷%張俊霞%崔立文%張慧然
리동묘%서금승%풍우%장성뢰%장준하%최립문%장혜연
维拉帕米%肌细胞,平滑肌%血管钙化%钙通道
維拉帕米%肌細胞,平滑肌%血管鈣化%鈣通道
유랍파미%기세포,평활기%혈관개화%개통도
Verapamil%Myocytes,smooth muscle%Vascular calcification%Calcium channels
目的：探讨维拉帕米抑制高磷诱导大鼠血管平滑肌细胞（ VSMCs）钙化的作用及可能机制。方法2014年12月—2015年6月选取5～8周龄清洁级健康雄性SD大鼠6只，体外分离培养大鼠VSMCs，采用免疫组化法鉴定。将对数期VSMCs随机分为正常对照组〔含10%胎牛血清（ FBS）培养基〕、高磷组（高磷培养基，含10 mml/Lβ-甘油磷酸）、维拉帕米干预组（在高磷培养基的基础上加入维拉帕米至浓度为20 mmol/L）。细胞接受2、4、6 d干预后，检测VSMCs内钙离子水平，采用反转录-聚合酶链反应（ RT-PCR）检测 VSMCs 内目的基因（ smad1、runx2 mRNA）表达水平。细胞接受14 d干预后进行钙化检测，包括钙水平测定和茜素红染色情况。结果高磷组、维拉帕米干预组2、4、6 d后VSMCs内钙离子水平均高于正常对照组（ P<0.05）；维拉帕米干预组2、4、6 d后VSMCs内钙离子水平均低于高磷组（ P<0.05）。高磷组、维拉帕米干预组4、6 d后VSMCs内钙离子水平分别高于2 d后（ P<0.05）；高磷组6 d后VSMCs内钙离子水平高于4 d后（P<0.05）；维拉帕米干预组6 d后VSMCs内钙离子水平低于4 d后（P<0.05）。高磷组、维拉帕米干预组2、4、6 d后VSMCs内smad1、runx2 mRNA表达水平均高于正常对照组（P<0.05）；维拉帕米干预组2、4、6 d后VSMCs内smad1、runx2 mRNA表达水平均低于高磷组（P<0.05）。高磷组4、6 d后VSMCs内smad1、runx2 mRNA表达水平分别高于2 d后，6 d后VSMCs内smad1、runx2 mRNA表达水平分别高于4 d后（P<0.05）；维拉帕米干预组4 d后VSMCs内smad1 mRNA表达水平高于2 d后，runx2 mRNA表达水平低于2 d后，6 d 后 VSMCs内 smad1、runx2 mRNA 表达水平均高于2、4 d 后（ P <0.05）。高磷组、维拉帕米干预组VSMCs钙水平均高于正常对照组（P<0.05）；维拉帕米干预组VSMCs钙水平低于高磷组（P<0.05）。高磷组、维拉帕米干预组橘红色钙化结节较正常对照组多，维拉帕米干预组橘红色钙化结节较高磷组少。结论维拉帕米在高磷诱导的VSMCs钙化中起抑制作用，其可能是通过抑制钙离子内流进而抑制smad1表达，进一步抑制runx2表达，进而抑制VSMCs发生表型转化来实现的。
目的：探討維拉帕米抑製高燐誘導大鼠血管平滑肌細胞（ VSMCs）鈣化的作用及可能機製。方法2014年12月—2015年6月選取5～8週齡清潔級健康雄性SD大鼠6隻，體外分離培養大鼠VSMCs，採用免疫組化法鑒定。將對數期VSMCs隨機分為正常對照組〔含10%胎牛血清（ FBS）培養基〕、高燐組（高燐培養基，含10 mml/Lβ-甘油燐痠）、維拉帕米榦預組（在高燐培養基的基礎上加入維拉帕米至濃度為20 mmol/L）。細胞接受2、4、6 d榦預後，檢測VSMCs內鈣離子水平，採用反轉錄-聚閤酶鏈反應（ RT-PCR）檢測 VSMCs 內目的基因（ smad1、runx2 mRNA）錶達水平。細胞接受14 d榦預後進行鈣化檢測，包括鈣水平測定和茜素紅染色情況。結果高燐組、維拉帕米榦預組2、4、6 d後VSMCs內鈣離子水平均高于正常對照組（ P<0.05）；維拉帕米榦預組2、4、6 d後VSMCs內鈣離子水平均低于高燐組（ P<0.05）。高燐組、維拉帕米榦預組4、6 d後VSMCs內鈣離子水平分彆高于2 d後（ P<0.05）；高燐組6 d後VSMCs內鈣離子水平高于4 d後（P<0.05）；維拉帕米榦預組6 d後VSMCs內鈣離子水平低于4 d後（P<0.05）。高燐組、維拉帕米榦預組2、4、6 d後VSMCs內smad1、runx2 mRNA錶達水平均高于正常對照組（P<0.05）；維拉帕米榦預組2、4、6 d後VSMCs內smad1、runx2 mRNA錶達水平均低于高燐組（P<0.05）。高燐組4、6 d後VSMCs內smad1、runx2 mRNA錶達水平分彆高于2 d後，6 d後VSMCs內smad1、runx2 mRNA錶達水平分彆高于4 d後（P<0.05）；維拉帕米榦預組4 d後VSMCs內smad1 mRNA錶達水平高于2 d後，runx2 mRNA錶達水平低于2 d後，6 d 後 VSMCs內 smad1、runx2 mRNA 錶達水平均高于2、4 d 後（ P <0.05）。高燐組、維拉帕米榦預組VSMCs鈣水平均高于正常對照組（P<0.05）；維拉帕米榦預組VSMCs鈣水平低于高燐組（P<0.05）。高燐組、維拉帕米榦預組橘紅色鈣化結節較正常對照組多，維拉帕米榦預組橘紅色鈣化結節較高燐組少。結論維拉帕米在高燐誘導的VSMCs鈣化中起抑製作用，其可能是通過抑製鈣離子內流進而抑製smad1錶達，進一步抑製runx2錶達，進而抑製VSMCs髮生錶型轉化來實現的。
목적：탐토유랍파미억제고린유도대서혈관평활기세포（ VSMCs）개화적작용급가능궤제。방법2014년12월—2015년6월선취5～8주령청길급건강웅성SD대서6지，체외분리배양대서VSMCs，채용면역조화법감정。장대수기VSMCs수궤분위정상대조조〔함10%태우혈청（ FBS）배양기〕、고린조（고린배양기，함10 mml/Lβ-감유린산）、유랍파미간예조（재고린배양기적기출상가입유랍파미지농도위20 mmol/L）。세포접수2、4、6 d간예후，검측VSMCs내개리자수평，채용반전록-취합매련반응（ RT-PCR）검측 VSMCs 내목적기인（ smad1、runx2 mRNA）표체수평。세포접수14 d간예후진행개화검측，포괄개수평측정화천소홍염색정황。결과고린조、유랍파미간예조2、4、6 d후VSMCs내개리자수평균고우정상대조조（ P<0.05）；유랍파미간예조2、4、6 d후VSMCs내개리자수평균저우고린조（ P<0.05）。고린조、유랍파미간예조4、6 d후VSMCs내개리자수평분별고우2 d후（ P<0.05）；고린조6 d후VSMCs내개리자수평고우4 d후（P<0.05）；유랍파미간예조6 d후VSMCs내개리자수평저우4 d후（P<0.05）。고린조、유랍파미간예조2、4、6 d후VSMCs내smad1、runx2 mRNA표체수평균고우정상대조조（P<0.05）；유랍파미간예조2、4、6 d후VSMCs내smad1、runx2 mRNA표체수평균저우고린조（P<0.05）。고린조4、6 d후VSMCs내smad1、runx2 mRNA표체수평분별고우2 d후，6 d후VSMCs내smad1、runx2 mRNA표체수평분별고우4 d후（P<0.05）；유랍파미간예조4 d후VSMCs내smad1 mRNA표체수평고우2 d후，runx2 mRNA표체수평저우2 d후，6 d 후 VSMCs내 smad1、runx2 mRNA 표체수평균고우2、4 d 후（ P <0.05）。고린조、유랍파미간예조VSMCs개수평균고우정상대조조（P<0.05）；유랍파미간예조VSMCs개수평저우고린조（P<0.05）。고린조、유랍파미간예조귤홍색개화결절교정상대조조다，유랍파미간예조귤홍색개화결절교고린조소。결론유랍파미재고린유도적VSMCs개화중기억제작용，기가능시통과억제개리자내류진이억제smad1표체，진일보억제runx2표체，진이억제VSMCs발생표형전화래실현적。
Objective To explore the effect and mechanism of verapamil on the calcification of vascular smooth muscle cells(VSMCs)induced by hyperphosphate. Methods From December 2014 to June 2015,selecte 6 healthy male SD rats of 5 to 8 weeks and cleaning grade vascular smooth muscle cells were cultured in vitro and were determined by immunohistochemistry. The VSMCs in logarithmic phrase were randomly divided into normal control group ( 10% FBS culture medium),hyperphosphate group( hyperphosphate culture medium)and verapamil group( hyperphosphate culture medium+20 nmol/L verapamil). Deteation of intracelluar calcium ion level of VSMCs after culture for 2,4 and 6 days. RT-PCR was used to observe the expression of VSMCs smad1 and runx2 mRNA after culture for 2,4,6 days. After culture for 14 days, calcification test was conducted,including calcium level measurement and alizarin red staining. Results After culture for 2,4 and 6 days,the hyperphosphate group and verapamil group were higher(P<0. 05)than normal control group in calcium ion level of VSMCs;after culture for 2,4 and 6 days,verapamil group was lower(P <0. 05)than hyperphosphate group in calcium ion level of VSMCs. After culture for 4 and 6 days,hyperphosphate group and verapamil group had higher(P<0. 05) calcium ion level of VSMCs than that after culture for 2 days;hyperphosphate group had higher(P<0. 05)calcium ion level of VSMCs after culture for 6 days than that after culture for 4 days;verapamil group had lower ( P<0. 05 )calcium ion level of VSMCs after culture for 6 days than that after culture for 4 days. After culture for 2 ,4 and 6 days hyperphosphate group and verapamil group were higher(P<0. 05)than normal control group in the expression of VSMCs smad1 and runx2 mRNA;after culture for 2,4 and 6 days,verapamil group was lower(P <0. 05)than hyperphosphate group in the expression of VSMCs smad1 and runx2 mRNA. Hyperphosphate group had higher ( P <0. 05 ) expression of VSMCs smad1 and runx2 mRNA after culture for 4 days and 6 days than that after culture for 2 days and had higher(P<0. 05)expression of VSMCs smad1 and runx2 mRNA after culture for 6 days than that after culture for 4 days;verapamil group had higher( P<0. 05 )expression of VSMCs smad1 mRNA and lower(P<0. 05)expression of VSMCs runx2 mRNA after culture for 4 days than that after culture for 2 days;verapamil group had higher( P<0. 05 )expression of VSMCs smad1 and runx2 mRNA after culture for 6 days than that after culture for 2 days and 4 days. Hyperphosphate group and verapamil group had higher ( P<0. 05 )calcium level of VSMCs than normal control group,verapamil group was lower(P<0. 05)than hyperphosphate group in calcium level. Hyperphosphate group and verapamil group had more orange calcification nodules than normal control group;verapamil group had less orange calcification nodules than hyperphosphate group. Conclusion Verapamil may protect against hyperphosphate - induced calcification of VSMCs and may inhibit VSMCs phenotypic transformation by downregulating smad1 expression and further inhibiting runx2 expression.
