【作者】 刘波；

【导师】 汤成春；

【作者基本信息】 东南大学 ， 内科学（心血管）， 2020， 博士

【摘要】 第一部分探讨低氧对TG2活性和表达的影响目的:探讨慢性低氧对TG2活性和表达的影响。方法:体外实验,利用组织贴壁法原代分离培养第3-4级人肺静脉平滑肌细胞（hPVSMC）,3-4代人肺静脉平滑肌细胞用作细胞实验,慢性低氧（1%O2）和常氧条件下,利用适时荧光定量PCR（qPCR）、蛋白免疫印迹技术（Western Blot）、（biotinamido）pentylamine（BP）法等方法检测转谷酰胺酶2（TG2）的表达、活性;在体实验,分离肺静脉组织利用适时荧光定量PCR（qPCR）、蛋白免疫印迹技术（WB）等方法检测TG2的表达;利用免疫组化技术观察TG2蛋白在肺血管定位表达情况。结果:经鉴定成功分离培养第3-4级人肺静脉平滑肌肌细胞,纯度高,细胞状态良好。慢性低氧可增强人肺静脉平滑肌细胞TG2 mRNA、蛋白质的表达和活性并呈时间依赖性;慢性低氧可增强小鼠肺静脉TG2的表达,TG2特异性敲除小鼠肺静脉上不表达TG2蛋白,TG2定位表达于肺血管平滑肌层。结论:慢性低氧可增加PVSMC上TG2活性及表达,并呈时间依赖性。第二部分探讨TG2调控5-HT化对SERCA2活性的影响及其破坏Ca²⁺平衡的机制目的:探讨慢性低氧下TG2调控5-HT化对SERCA2活性的影响,进一步以SOCE方式激活TRPC6通道促进钙离子内流的可能机制。方法:慢性低氧（1%O2）和常氧条件下利用重组腺病毒、干扰小RNA（siRNA）技术过表达和沉默TG2基因,运用免疫共沉淀方法（co-IP）、有机磷测定法检测肌内质网钙三磷酸腺苷酶2（SERCA2）的5-羟色胺化（s-SERCA2）表达水平和SERCA2的活性,并进一步运用钙离子成像技术检测人肺静脉平滑肌细胞内钙离子的变化。在体实验,分离肺静脉组织用co-IP方法检测肺静脉s-SERCA2的表达水平、有机磷测定法检测肺静脉SERCA2活性。运用qPCR、WB的方法检测人肺静脉平滑肌细胞经典瞬间受体电位阳离子通道1（TRPC1）和TRPC6的表达情况;慢性低氧条件下,分别沉默和过表达TRPC1基因和TRPC6基因,运用钙离子成像技术检测人肺静脉平滑肌细胞内钙离子浓度([Ca²⁺]i)和钙库操纵性钙离子内流（SOCE）。结果:体内体外慢性低氧可增强s-SERCA2的表达,受TG2调控。体外常氧下,TG2基因沉默时细胞产生更多的无机磷（IP）,TG2基因过表达时,细胞产生较少的无机磷;与常氧组相比,低氧下TG2基因沉默没有增加无机磷的产量。体内常氧和低氧下,Tgm2-/-小鼠比WT小鼠产生更多的无机磷,但是低氧下WT小鼠与Tgm2-/-小鼠之间的无机磷产量没有差异。低氧（1%O2）条件下,细胞基础[Ca²⁺]i从（6.81±1.14）显着增加到（13.88±0.26）。常氧条件下,TG2基因沉默时细胞基础[Ca²⁺]i从（6.81±1.14）降至（1.40±0.04）,TG2基因过表达时细胞基础[Ca²⁺]i升至（8.30±0.03）;低氧条件下,TG2基因沉默时细胞基础[Ca²⁺]i从（13.88±0.26）降至（6.95±0.16）,TG2基因过表达时细胞基础[Ca²⁺]i升至（20.48±0.25）。低氧促进正常细胞荧光峰值从（164.33±7.64）显着增加到（228.00±14.42）;常氧条件下,TG2基因沉默时荧光峰值从（164.33±7.64）降至（130.33±4.51）,TG2基因过表达时荧光峰值升至（207.00±6.25）;低氧条件下,TG2基因沉默时,细胞荧光峰值从（228.00±14.42）降至（165.33±11.01）,当TG2基因过表达时荧光峰值升至（383.67±13.50）。低氧时TRPC6 mRNA和蛋白的表达显著增加且呈时间依赖性,而TRPC1 mRNA和蛋白的表达没有显著增加或减少;低氧下,过表达TRPC6基因时基础[Ca²⁺]i从（2.81±0.29）增加到（5.04±0.05）,沉默TRPC6基因时减少到（1.64±0.06）,沉默和过表达TRPC1基因时基础[Ca²⁺]i分别为（2.82±0.25）和（2.73±0.29）,过表达TRPC6基因时,荧光峰值从（193.00±4.00）增加到（314.67±2.89）,沉默TRPC6基因时减少到（114.33±2.31）,沉默和过表达TRPC1基因时荧光峰值分别为（200.67±10.60）和（206.00±5.00）。结论:低氧可促进TG2介导的SERCA2的5-羟色胺化,SERCA2的5-强色胺化抑制其活性,促进钙离子内流;慢性低氧可激活h PVSMC TRPC6介导的SOCE促进细胞外钙内流。第三部分SERCA2活性改变影响人肺静脉平滑细胞生物学行为的分子及钙信号机制研究目的:探讨SERCA2的活性对h PVSMCs增殖、凋亡、迁移及细胞内钙离子浓度的影响方法:使用SERCA2激动剂（PST-2744）和抑制剂（CPA）干预hPVSMC,采用CCK-8、流式细胞技术、划痕实验、qPCR、Western blot、钙离子成像等技术,检测细胞增殖与凋亡、细胞迁移、分化和去分化表型标志蛋白表达、[Ca²⁺]i等变化。结果:CPA抑制细胞凋亡,促进细胞增殖和迁移,而PST-2744促进细胞凋亡,抑制细胞增殖和迁移;CPA促进细胞从分化表型转变为去分化表型,PST-2744促进细胞从去分化表型转化为分化表型。正常细胞[Ca²⁺]i为（6.97±1.16）,用CPA处理12小时和24小时的细胞基础[Ca²⁺]i分别为（8.69±0.43）和（14.82±0.88）。用PST-2744处理12小时和24小时的细胞的基础[Ca²⁺]i分别为（5.36±0.46）和（4.03±0.19）;正常荧光峰值为（171.33±6.11）,用CPA处理12小时和24小时的细胞的荧光峰值荧光分别为（221.67±8.62）和（265.00±6.00）,用PST-2744处理12小时和24小时的细胞荧光峰值为（143.67±5.69）和（116.67±6.51）。结论:CPA抑制细胞凋亡,促进细胞增殖和迁移,而PST-2744促进细胞凋亡,抑制细胞增殖和迁移;CPA可促进细胞外钙内流,PS-2744降低细胞外钙内流,并呈时间依赖性;CPA促进从分化表型转变为去分化表型,PST-2744促进细胞从去分化表型转化为分化表型。第四部分整体水平探讨5-HT化在低氧性肺静脉重塑中的作用目的:探讨血管平滑肌TG2特异性敲除小鼠5-HT化在低氧性肺静脉重塑中的作用方法:采用的经典的慢性低氧干预方法,建立5-HT化关键调控蛋白TG2敲除（Tgm2-/-）小鼠和野生型C57BL/6J小鼠（WT）的慢性低氧性肺高血压（HPH）模型,测定小鼠右心室收缩压（RVSP）,分离右心室（RV）、左心室加室间隔（LV+S）并依次称重计算两者的比值[RV/（LV+S）];HE染色检测肺血管壁厚度与血管外径比值（MT%）、血管壁面积与血管总面积比值（MA%）。结果:低氧增加WT小鼠肺静脉s-SERCA2蛋白表达及SERCA2活性,对Tgm2-/-小鼠肺静脉s-SERCA2蛋白表达及SERCA2活性无影响;常氧条件下,Tgm2-/-小鼠和WT小鼠的RVSP分别为（18.15±0.45）mm Hg和（18.35±0.76）mm Hg;低氧6周后,WT小鼠RVSP从（18.35±0.76）mm Hg增加到（34.05±0.99）mm Hg,Tgm2-/-小鼠RVSP从（18.15±0.45）mm Hg增加到（22.79±6.79）mm Hg;常氧条件下,Tgm2-/-小鼠和WT小鼠的RVHI分别为（0.20±0.010）和（0.21+0.009）,低氧6周后,Tgm2-/-小鼠和WT小鼠的RVHI分别为（0.25±0.063）和（0.34+0.055）;常氧条件下,Tgm2-/-小鼠和WT小鼠的MT%分别为（9.90±0.30）和（10.37±0.67）,MA%分别为（19.13±1.00）和（18.40±0.62）;低氧条件下,Tgm2-/-小鼠和WT小鼠的MT%分别为（9.63±0.70）和（43.30±1.67）,MA%分别为（18.97±0.47）和（70.67±0.50）。结论:敲除TG2基因小鼠可逆转慢性低氧性肺动脉压力升高及肺血管重塑。更多还原

【Abstract】 PartⅠ.Effects of Hypoxia on TG2 Activity and ExpressionObject:To investigate effects of chronic hypoxia on TG2 activity and expression.Methods:Human venous smooth muscle cells（hPVSMC）were isolated and cultured from the intrapulmonary veins(4^th level)by tissue explants adherence method.hPVSMCs were identified by positive immunostaining with an anti-α-SMA monoclonal antibody.qPCR,Western blotting（WB）and（biotinamido）pentylamine（BP）were used to detect expression and activity of transglutaminase 2（TG2）under chronic hypoxia（1%O2）and normoxia.Immunohistochemistry was used to detect location of TG2 protein on pulmonary vessels.qPCR and Western blotting（WB）were used to detect expression of TG2 on pulmonary vessels.Results:3th-4th generation hPVSMCs were used for cell experiments.It was identified that 3-4th human pulmonary vein smooth muscle cells were successfully isolated.Cells were high purity and in good condition,and cells can be used in experiments.Chronic hypoxia enhanced TG2 expression and activity in hPVSMC,and was time-dependent.Chronic hypoxia enhanced TG2 expression in the pulmonary veins of mice.TG2localized on pulmonary smooth muscle layer.Conclusion:Hypoxia enhanced TG2 expression and activity,and TG2 localized on pulmonary smooth muscle layer.Part Ⅱ.To investigate the mechanisms of TG2-mediated SERCA2 Serotonination and disruption Ca²⁺ balanceObject: To investigate mechanism of TG2-mediated SERCA2 Serotonination and hypoxia promoted calcium influx through TRPC6-mediated SOCE.Methods: siRNA and adenovirus were used to silence and overexpress the interest gene.co-Immunoprecipitation（co-IP）and micro-Ca²⁺ ATP enzyme kit were used to detect sSERCA2 expression and SERCA2 activity when the TG2 gene silenced oroverexpressed under normoxia and hypoxia.Intracellular calcium ion was measured by using Fluo-4AM probe under normoxia and hypoxia.q PCR and WB were used to detect TRPC1 and TRPC6 expression on hPVSMCs under normoxia and hypoxia.Fluo-4AM probe was used to detect basal [Ca²⁺]i and SOCE when the TRPC1 and TRPC6 gene silenced and overexpressed under hypoxia.Results: Cells produced more inorganic phosphorus when the TG2 gene was silenced,whereas cells produced less organic phosphorus when the TG2 gene overexpressed under normoxia.Hypoxia significantly prevented cells from producing inorganic phosphorus,which was regulated by TG2.When compared to the normoxia group,silencing of the TG2 gene did not increase the production of inorganic phosphorus under hypoxia.Expression of s-SERCA2 protein was significantly increased when the TG2 gene was overexpressed,whereas the expression of s-SERCA2 protein decreased when the TG2 gene was silenced under normoxia.When compared to the normoxia group,the expression of s-SERCA2 protein significantly increased under hypoxia,however the expression did not significantly increase when the TG2 gene was silenced under hypoxia.Hypoxia（1% O2）induced a marked increase in basal [Ca²⁺]i from 6.81±1.14 to 13.88±0.26.In the normoxia group,the cell basal [Ca²⁺]i decreased from 6.81±1.14 to 1.40±0.04 when the TG2 gene was silenced,and increased from 6.81±1.14 to 8.30±0.03 when the TG2 gene was overexpressed.In the hypoxia group,the cell basal [Ca²⁺]i decreased from 13.88±0.26 to 6.95±0.16 when the TG2 gene was silenced,and increased to 20.48±0.25 when the TG2 gene was overexpressed.Hypoxia induced a marked increase in the peak of time-Δfluorescence curve from 164.33±7.64 to 228.00±14.42.In the normoxia group,the peak Δ[Ca²⁺]i decreased from 164.33±7.64 to 130.33±4.51 when the TG2 gene was silenced,and increased to 207.00±6.25 when the TG2 gene was overexpressed.In addition,in the hypoxia group,the peak Δ[Ca²⁺]i decreased from 228.00±14.42 to 165.33±11.01 when the TG2 gene was silenced,and increased to 383.67±13.50 when the TG2 gene was overexpressed.Hypoxia increased expression of TRPC6,not TRPC1,on hPVSMCs.The basal [Ca²⁺]i increased from 2.81±0.29 to 5.04±0.05 after TRPC6 gene overexpression,but decreased to 1.64±0.06 after TRPC6 gene silencing.The basal [Ca²⁺]i of silencing and overexpressing of theTRPC1 gene were 2.82±0.25 and 2.73±0.29.The peak fluorescence increased from 193.00±4.00 to 314.67±2.89 after TRPC6 gene overexpression,and decreased to 114.33±2.31 after TRPC6 gene silencing.The peak fluorescence of silencing and overexpressing TRPC1 gene were 200.67 ±10.60 and 206.00±5.00.Conclusion: Hypoxia activated TRPC6-mediated SOCE to promote the extracellular calcium influx.Hypoxia promoted TG2-mediated s-SERCA2,and s-SERCA2 inhibited activity of SERCA2 and promoted calcium influx through TRPC6-mediated SOCE.Part Ⅲ.Effect of SERCA2 activity on cell biological behavior and calcium signalingObject: To investigate the effects of SERCA2 activity on proliferation,apoptosis,migration and intracellular calcium in hPVSMCsMethods: Cells were treated with CPA（SERCA2 inhibitor）and PST-2744（SERCA2 agonist）for 12 and 24 hours,respectively.CCK-8,flow cytometry,wound scratch,q PCR,WB and Fluo-4AM probe were used to detect hPVSMCs apoptosis,proliferation,cell migration,cell phenotypic protein expression and [Ca²⁺]i,respectively.Results: CPA inhibited cell apoptosis,and promoted cell proliferation and migration,whereas PST-2744 promoted cell apoptosis,and inhibited cell proliferation and migration in a time-dependent manner.CPA promoted transformation from a differentiated phenotype to a dedifferentiated phenotype,and PST-2744 promoted transformation from a dedifferentiated phenotype to a differentiated phenotype.Normal [Ca²⁺]i was 6.97±1.16,the [Ca²⁺]i of cells treated with CPA for 12 hours and 24 hours was 8.69±0.43 and 14.82±0.88.In addition,the [Ca²⁺]i of cells treated with PST-2744 for 12 hours and 24 hours was 5.36±0.46 and 4.03±0.19.The normal peak fluorescence was 171.33±6.11.The peak fluorescence of cells treated with CPA for 12 hours and 24 hours were 221.67±8.62 and 265.00±6.00,and the peak fluorescence the of cells treated with PST-2744 for 12 hours and 24 hours was 143.67±5.69 and 116.67±6.51.Conclusion: SERCA2 activity played an important role in regulating hPVSMCs proliferation,apoptosis,migration,cell phenotype and calcium ion influx.Part Ⅳ.Role of SERCA2 serotonination on hypoxic pulmonary vein remodeling in vivoObject: Establishing PH model to investigate role of SERCA2 serotonination on hypoxic pulmonary vein remodelingMethods: Wild-type（WT）mice（n=6）and TG2 knockout（Tgm2-/-）mice（n=6）were exposed to hypoxia（10% O2）in a normobaric chamber for 6 weeks.Another set of identical WT mice（n=6）and Tgm2-/-mice（n=6）were kept under normal conditions for 6 weeks,and represented the normoxia groups.co-IP and micro-Ca²⁺ ATP enzyme kit were used to detect s-SERCA2 and SERCA2 activity of isolated pulmonary veins.Closed-chest insertion into the right ventricle（RV）was used to detect RVSP.To evaluate the extent of RV hypertrophy,the weights of the RV and the left ventricle plus interventricular septum（LV+S）were measured separately.Hematoxylin-eosin（HE）staining and Image J Plus 6.0 software were used to detect percentage medial layer thickness（MT%=100 × [medial layer thickness]/[vessel semidiameter]）and area（MA%=100×[cross‐sectional vessel area]）of peripheral pulmonary vessels.Results: The RVSP of Tgm2-/-mice and WT mice was 18.15±0.45 mm Hg and 18.35+0.76 mm Hg under normoxia.The RVSP of WT mice increased from 18.15±0.45 mm Hg to 34.05±0.99 mm Hg after hypoxia exposure for 6 weeks.The RVSP of Tgm2-/-mice under hypoxia 22.79±6.79 mm Hg was slightly higher when compared to that of WT mice under normoxia.The RVHI of Tgm2-/-mice and WT mice were 0.20±0.010 and 0.21+0.009 under normoxia,respectively.Moreover,the RVHI of Tgm2-/-mice and WT mice were 0.25±0.063 and 0.34+0.055 under hypoxia.The MT% of Tgm2-/-mice and WT mice were 9.90±0.30 and 10.37±0.67,and MA% of Tgm2-/-mice and WT mice were19.13±1.00 and 18.40±0.62 under normoxia,and respectively.The MT% of Tgm2-/-mice and WT mice were 9.63±0.70 and 43.30±1.67,and MA% of Tgm2-/-mice and WT mice were8.97±0.47 and 70.67±0.50 under hypoxia,and respectively.Conclusion:TG2 was a key enzyme that regulateed SERCA2 serotonination in vivo,and knocking out the TG2 gene reversed pulmonary artery pressure and pulmonary vein remodeling under hypoxia.更多还原