摘要
目的研究人E1A激活基因阻遏子基因(CREG)蛋白在人血管平滑肌细胞迁移中的作用。方法构建含有myc和His标签的野生型CREG(wtCREG)和去糖基化突变型CREG(mCREG)真核表达载体pcDNA3.1myc-His/wt/mCREG。转染人293F细胞株,Ni-NTA亲合层析方法纯化获得wtCREG和mCREG蛋白;将重组wt-CREG(400 nmol/L)及mCREG蛋白(400 nmol/L),分别加入体外培养的低表达CREG的人血管平滑肌细胞OB2中,应用Western blot、细胞刮伤实验、明胶酶电泳方法观察两种重组人CREG蛋白对人血管平滑肌细胞迁移和分化等生物学行为的影响;通过不同浓度(2、4、8 mg/L)的胰岛素生长因子2受体(M6P/IGF2R)中和抗体与人M6P/IGF2R细胞外结构域蛋白小肽分别进行阻断实验,分析M6P/IGF2R是否参与介导CREG蛋白对平滑肌细胞迁移的调控。结果刮伤实验发现,加入最佳效应浓度的wtCREG和mCREG蛋白24 h后,OB2组细胞的迁移能力均明显下降;明胶酶电泳和Western blot检测结果也证实,两种重组CREG蛋白均可以使细胞外基质金属蛋白酶2、基质金属蛋白酶9的合成及活性减少,而组织金属蛋白酶抑制物表达则明显增加;同时,Western blot分析也证实,平滑肌细胞分化标志蛋白myocardin、SMα-actin、肌球蛋白重链和caldesmin表达增加,LM-1和FN表达减少。提示两种重组CREG蛋白均能够抑制体外培养的人血管平滑肌细胞迁移,促进其分化。IGF2R中和抗体和IGF2R小肽阻断实验证实:不同浓度的Anti-M6P/IGF2R能有效阻断两种CREG蛋白对人血管平滑肌细胞迁移和细胞外基质合成的调控作用。并且,M6P/IGF2R的第11～13结构域小肽片段对wt/mCREG蛋白的生物学效应也有明显的阻断作用。结论重组CREG蛋白可能通过细胞膜表面M6P/IGF2R的11～13结构域抑制人血管平滑肌细胞迁移和细胞外基质分泌,维持细胞分化。
Aim The present study aimed to investigate the bio-function and mechanism of cellular repressor of E1A-stimulated genes （CREG） protein on the migration of vascular smooth muscle cells （SMCs）. Methods Human wild-type and glycosylation mutant CREG proteins （named wtCREG or mCREG） were transfected and purified in human 293F cells. Human SMCs with CREG knocked-down expression （named OB2） were used to evaluate the effects of two kinds of recombinant CREG protein. The migration of OB2 cells was evaluated by wound-healing assay. The expression and activity was detected by Western blot and gelatin zymography. The differentiational marker proteins of SMCs were identified to express by Western blot analysis. Furthermore, using soluble mannose-6-phosphate/insulin-like growth factor-2 receptor （M6P/IGF2R） fragments and M6P/IGF2R neutralizing antibody, the blocking analysis was finished by detecting the migration of OB2 cells. Results Both wtCREG and mCREG （400 mnol/L） inhibit the migration of OB2 cells. Mean- while, the expression and activity of matrix metalloproteinase-2 （MMP2） and matrix metalloproteinase-9 （MMP9） were iden- tified to reduce in OB2 ceils when treated with recombinant CREG protein. Reversely, the expressions of myocardin, smooth muscle ot-actin （ SM o^-actin）, myosin heavy chain （MHC） and caldesmin were detected to enhance obviously. Fur- ther blocking experiments using soluble M6P/IGF2R fragments and M6P/IGF2R neutralizing antibody suggest that two kinds of recombinant protein are adequate for modulating SMC migration by binding to domains 11-13. Conclusion These data suggest that soluble CREG protein can exert its biological function via binding to cell surface M6P/IGF2R, and thereby pro- vide novel insights into CREG modulation of SMC phenotypic switching from contractile to migration.
出处
《中国动脉硬化杂志》
CAS
CSCD
北大核心
2012年第11期961-967,共7页
Chinese Journal of Arteriosclerosis
基金
国家自然科学基金资助项目(81070097
30971218)
辽宁省科技攻关项目(200925009-9)
辽宁省自然科学基金资助项目(20092088
20092090
20091100)
