经穴注射BMSCs联合益气活血方对DM后肢缺血大鼠血管再生作用机制研究
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摘要
目的：研究益气活血方血清对骨髓间充质干细胞(Bone Mesenchymal Stem Cells, BMSCs)增殖活力的影响,以及经穴注射BMSCs联合益气活血中药促进糖尿病大鼠后肢缺血血管再生的机制,为临床中医血管外科防治下肢动脉缺血性疾病提供新的思路和方法。
方法：(一)体外实验：采用全细胞贴壁培养法纯化BMSCs,用流式细胞术鉴定BMSCs。采集空白对照大鼠、益气药、活血药、益气活血方灌胃大鼠的含药血清,应用MTT法检测10%浓度以上四种不同含药血清对第3代BMSCs增殖活力的影响；以及5%、10%、20%不同浓度空白血清与益气活血含药血清对第3代BMSCs增殖活力的影响；(二)体内实验：80只SD大鼠随机分为8组：正常血糖假手术组、糖尿病大鼠假手术组、糖尿病大鼠缺血组、益气活血中药治疗组、局部注射BMSCs组、局部注射BMSCs+益气活血方治疗组、穴位注射BMSCs组、穴位注射BMSCs+益气活血方治疗组,每组10只。
DM模型：采用空腹腹腔注射50.0mg/Kg的STZ；缺血模型：剥离切除DM大鼠左后肢股动脉和所有侧支血管。于术后2、7、14、21天进行大鼠一般行为学观察,记录血糖水平,体重等；应用PeriScan PIM型激光多普勒血流成像仪,比较各组间大鼠后肢血流变化；免疫组织化学法检测肌动蛋白(α-actin)、血小板内皮细胞粘附分子(platelet endothelial cell adhesion molecule-1, PEC AM-1/CD31、血管性血友病因子(von willebrand factor,vWF)标记的血管密度；免疫组织化学检测血管内皮细胞生长因子(vascular endothelial cell growth factor, VEGF)、碱性成纤维细胞生长因子(basic fibroblast growth factor, bFGF)在肌肉组织中的表达量；(三)用ELISA去检测益气活血中药对后肢缺血大鼠血清VEGF、G-CSF、SDF-1浓度的影响。
结果：(一)体外实验：采用全骨髓贴壁法获取的BMSCs形态多为椭圆或长梭形,呈旋涡状生长。流式细胞术检测第三代BMSCs,CD44、CD106呈阳性表达,基本不表达CD45、CD34.(1)不同方含药血清培养BMSCs,在第2、4、5天细胞快速增长,均较前一天增长明显,第6天时活血方血清组与益气活血方血清组细胞增长已呈下降趋势,其他三组在第6天细胞增殖达最大,第7天时呈下降趋势。BMSCs去除基线增殖率组间比较,培养第2天时,各组间比较虽无差异,但活血方血清组达最大；第4天时,益气活血方组达最大,活血方血清组次之；培养第6天时,益气方血清组较其他三组大,益气活血方血清组次之,活血方血清组最低；(2)在不同浓度含药血清培养下,10%益气活血方血清培养BMSCs从第2天起呈快速增长,去除基线增殖率达最大(69.95±7.50%),而20%空白血清、20%益气活血血清组对BMSCs增殖活力的影响仅高于10%FBS组。(二)体内实验：在本实验中,本方法制造糖尿病模型,成模率为87.14%,成模率高,成模稳定；采用结扎并切除DM大鼠股动脉及其侧支血管的方法制造后肢缺血模型,存活率为88.52%。(1)一般行为学观察：缺血造模后的大鼠状态萎靡,术后患肢冰凉,颜色苍白,甚至紫暗,多数出现肢体跛行、溃疡；剥离切除DM大鼠左后肢股动脉组大鼠PUI值明显低于假手术组,造模成功；(2)各组PUI差值比较,穴位+中药组PUI差值大幅增加,缺血后肢情况改善最明显,其次为穴位注射组、局部+中药组,中药组与局部注射组改善相对较小；(3)各组术侧与健侧后肢a-actin、CD31表达量比较均有所升高；穴位注射BMSCs+中药组后肢缺血DM大鼠术侧骨骼肌中VEGF mRNA、bFGF mRNA表达量明显升高、a-actin、CD31表达量平均光密度较其他组最高,而vWF的表达下降最明显；(三)注射BMSCs缺血组、BMSCs联合益气活血中药组大鼠血清中VEGF,SDF-1, G-CSF质量浓度较其他组显著升高。
结论：全细胞贴壁培养的大鼠BMSCs在细胞形态与表形表达上均具备BMSCs特征。(1)活血方血清对BMSCs增殖影响出现最早,益气方血清对BMSCs增殖影响持续时间最长,益气活血全方含药血清对培养BMSCs的体外增殖活力影响最大；10%益气活血中药含药血清可明显提高BMSCs的体外增殖能力；(2)经穴注射BMSCs联合益气活血中药能显著改善缺血DM大鼠后肢的血流灌注,可促进DM大鼠后肢缺血骨骼肌VEGF、 bFGF表达增加,血管内皮细胞与血管平滑肌细胞的增生,对预防动脉硬化有一定作用。(3)经穴注射BMSCs联合益气活血中药可明显促进大鼠血清中VEGF、SDF-1、G-CSF的表达。
Objective:
To study the influence of Qi-tonifying and Blood-activating Chinese herb (YQHX) serum on the proliferous ability of bone mesenchymal stem cells (BMSCs) and angiogenesis mechanism of ischemic vessel on diabetic rats with hind limbs ischemia by injection BMSCs and administering YQHX herb, thereby providing new understandings and methods for preventing and treating lower limb arterial ischemic diseases.
Methods:(1) In vitro, BMSCs was purified by all cells adherent culture method, which identified by flow cytometry. The serum was extracted from the rats in control group and the rats were given Qi-tonifying (YQ) herbs and Blood-activating (HX) herbs by intragastric administration. MTT assay was used to detect the influence of10%concentration of4different drug serums on the proliferous ability of the third generation of BMSCs and to detect the influence of5%,10%and20%concentration of control and Yiqihuoxue serum on the proliferous ability of the third generation of BMSCs.
(2) In vivo,80Sprague Dawley (SD) rats were randomly divided into8groups:normal blood sugar control (NMC) group, diabetes control (DMC) group, diabetes ischemic (DMI) group, Yiqihuoxue (YQHX) group, local injection BMSCs (L-BMSCs) group, L-BMSCs+YQHX group, acupoint injection BMSCS (Acu-BMSCs) group, and Acu-BMSCs+YQHX group,10rats per group. Establishing animal model of diabetic mellitus:50mg/kg of Streptozotocin (STZ) per rat was injected intraperitoneally. Ischemic model:arteria femoralis as well as all of collateral artery in the left hind limb of the rats were excised. The general state, level of blood sugar and weight were observed at the2nd,7th,14th and21th day after modeling. The change of blood flow in hind limb of the rats was detected by Laser Doppler Perfusion Imager (PeriScan PIM). Immunohisto-chemistry was used to detect the vascular density which signed by a-actin, platelet endothelial cell adhesion molecule-1(PECAM-1/CD31), and von Willebrand factor (vWF), and the expression of vascular endothelial cell growth factor (VEGF) and basic fibroblast growth factor (bFGF) in muscular tissue. Elisa method was used to detect the serum level of VEGF, G-CSF and SDF-1.
Results:Most of the BMSCs obtained by all cells adherent culture method was elliptical and long-fusiform shape, and grow spirally in vitro. The membranes of third generation of BMSCs were positive for CD44and CD106and negative for CD45and CD34detected by flow cytometry. BMSCs cultured by various medicated serum increased significantly on the2nd,4th and5th day compared with the previous day. On the6th day, BMSCs proliferation by HX serum and YQHX serum began to decrease, on the other hand, the other three groups reached a peak proliferation, and decreased on the7th day. There was no significant difference among each group on the2nd day, but BMSCs cultured by HX serum increased the most. On the4th day, BMSCs cultured by YQHX serum was highest of all groups, and HX serum group was the second. On the6th day, BMSCs YQHX group was second then followed by HX group which was still higher than the control group.(2) Among different concentration of medicated serum, only10%YQHX serum cultured BMSCs began to increased significantly on the2nd day. BMSCs cultured by20%control and YQHX serum were higher than the10%FBS group. In vivo, there was87.14%rats induced diabetes. The DM rat model rat of DM was high and stable. The survival rate of ischemic rat model established by excising arterial femoral is as well as all of collateral artery in the left hind limb was88.52%in DM rats.(1) Ethological results:the rat models exhibited cachexia, pallid and pale limb, even turning dark purple, claudication and ulcer in some. The PUI of the DM rats was lower than the control group.
(2) The PUI of ischemic hind limb of the rats in the Acu-BMSCs+YQHX group increased most obviously, second were Acu-BMSCs and L-BMSCs+YQHX group, and lastly the YQHX and Acu-BMSCs group.(3) The expression of a-actin and CD31in the ischemic limb were higher than the healthy limb in each group. The expression of VEGF and bFGF mRNA, a-actin and CD31increased, and VWF decreased in the Acu-BMSCs+YQHX group compared with other groups. The levels of VEGF, SDF-1and G-CSF in rat serum tested by ELISA of Acu-BMSCs and Acu-BMSCs+group were higher than other groups.
Conclusion:The rat BMSCs cultured by all cells adherent culture method has features of BMSCs in cellular morphology and phenotypic expression.(1) The influence of HX serum on proliferation capacity of BMSCs appeared earliest of all. The influence of YQ serum on proliferation activation of BMSCs continued longest of all. The influence of YQHX serum on proliferous capacity of BMSCs was biggest of all. In vitro, the proliferous capacity of BMSCs was markedly increased obviously by10%concentration of YQHXsemn.(2) The blood flow of the ischemic hind limb of the DM rats improved efficiency and the expression of VEGF and bFGF increased by injecting BMSCs and YQHX medicine at acupuncture points. It also enhances the hyperplasia of vascular endothelial cells and vascular smooth muscle cells and prevents atherosclerosis.(3)The expression of VEGF, SDF-1and G-SGF in serum was increased in injection BMSCs group and Acu-BMSCs+YQHX medicine group.
方法：(一)体外实验：采用全细胞贴壁培养法纯化BMSCs,用流式细胞术鉴定BMSCs。采集空白对照大鼠、益气药、活血药、益气活血方灌胃大鼠的含药血清,应用MTT法检测10%浓度以上四种不同含药血清对第3代BMSCs增殖活力的影响；以及5%、10%、20%不同浓度空白血清与益气活血含药血清对第3代BMSCs增殖活力的影响；(二)体内实验：80只SD大鼠随机分为8组：正常血糖假手术组、糖尿病大鼠假手术组、糖尿病大鼠缺血组、益气活血中药治疗组、局部注射BMSCs组、局部注射BMSCs+益气活血方治疗组、穴位注射BMSCs组、穴位注射BMSCs+益气活血方治疗组,每组10只。
DM模型：采用空腹腹腔注射50.0mg/Kg的STZ；缺血模型：剥离切除DM大鼠左后肢股动脉和所有侧支血管。于术后2、7、14、21天进行大鼠一般行为学观察,记录血糖水平,体重等；应用PeriScan PIM型激光多普勒血流成像仪,比较各组间大鼠后肢血流变化；免疫组织化学法检测肌动蛋白(α-actin)、血小板内皮细胞粘附分子(platelet endothelial cell adhesion molecule-1, PEC AM-1/CD31、血管性血友病因子(von willebrand factor,vWF)标记的血管密度；免疫组织化学检测血管内皮细胞生长因子(vascular endothelial cell growth factor, VEGF)、碱性成纤维细胞生长因子(basic fibroblast growth factor, bFGF)在肌肉组织中的表达量；(三)用ELISA去检测益气活血中药对后肢缺血大鼠血清VEGF、G-CSF、SDF-1浓度的影响。
结果：(一)体外实验：采用全骨髓贴壁法获取的BMSCs形态多为椭圆或长梭形,呈旋涡状生长。流式细胞术检测第三代BMSCs,CD44、CD106呈阳性表达,基本不表达CD45、CD34.(1)不同方含药血清培养BMSCs,在第2、4、5天细胞快速增长,均较前一天增长明显,第6天时活血方血清组与益气活血方血清组细胞增长已呈下降趋势,其他三组在第6天细胞增殖达最大,第7天时呈下降趋势。BMSCs去除基线增殖率组间比较,培养第2天时,各组间比较虽无差异,但活血方血清组达最大；第4天时,益气活血方组达最大,活血方血清组次之；培养第6天时,益气方血清组较其他三组大,益气活血方血清组次之,活血方血清组最低；(2)在不同浓度含药血清培养下,10%益气活血方血清培养BMSCs从第2天起呈快速增长,去除基线增殖率达最大(69.95±7.50%),而20%空白血清、20%益气活血血清组对BMSCs增殖活力的影响仅高于10%FBS组。(二)体内实验：在本实验中,本方法制造糖尿病模型,成模率为87.14%,成模率高,成模稳定；采用结扎并切除DM大鼠股动脉及其侧支血管的方法制造后肢缺血模型,存活率为88.52%。(1)一般行为学观察：缺血造模后的大鼠状态萎靡,术后患肢冰凉,颜色苍白,甚至紫暗,多数出现肢体跛行、溃疡；剥离切除DM大鼠左后肢股动脉组大鼠PUI值明显低于假手术组,造模成功；(2)各组PUI差值比较,穴位+中药组PUI差值大幅增加,缺血后肢情况改善最明显,其次为穴位注射组、局部+中药组,中药组与局部注射组改善相对较小；(3)各组术侧与健侧后肢a-actin、CD31表达量比较均有所升高；穴位注射BMSCs+中药组后肢缺血DM大鼠术侧骨骼肌中VEGF mRNA、bFGF mRNA表达量明显升高、a-actin、CD31表达量平均光密度较其他组最高,而vWF的表达下降最明显；(三)注射BMSCs缺血组、BMSCs联合益气活血中药组大鼠血清中VEGF,SDF-1, G-CSF质量浓度较其他组显著升高。
结论：全细胞贴壁培养的大鼠BMSCs在细胞形态与表形表达上均具备BMSCs特征。(1)活血方血清对BMSCs增殖影响出现最早,益气方血清对BMSCs增殖影响持续时间最长,益气活血全方含药血清对培养BMSCs的体外增殖活力影响最大；10%益气活血中药含药血清可明显提高BMSCs的体外增殖能力；(2)经穴注射BMSCs联合益气活血中药能显著改善缺血DM大鼠后肢的血流灌注,可促进DM大鼠后肢缺血骨骼肌VEGF、 bFGF表达增加,血管内皮细胞与血管平滑肌细胞的增生,对预防动脉硬化有一定作用。(3)经穴注射BMSCs联合益气活血中药可明显促进大鼠血清中VEGF、SDF-1、G-CSF的表达。
Objective:
To study the influence of Qi-tonifying and Blood-activating Chinese herb (YQHX) serum on the proliferous ability of bone mesenchymal stem cells (BMSCs) and angiogenesis mechanism of ischemic vessel on diabetic rats with hind limbs ischemia by injection BMSCs and administering YQHX herb, thereby providing new understandings and methods for preventing and treating lower limb arterial ischemic diseases.
Methods:(1) In vitro, BMSCs was purified by all cells adherent culture method, which identified by flow cytometry. The serum was extracted from the rats in control group and the rats were given Qi-tonifying (YQ) herbs and Blood-activating (HX) herbs by intragastric administration. MTT assay was used to detect the influence of10%concentration of4different drug serums on the proliferous ability of the third generation of BMSCs and to detect the influence of5%,10%and20%concentration of control and Yiqihuoxue serum on the proliferous ability of the third generation of BMSCs.
(2) In vivo,80Sprague Dawley (SD) rats were randomly divided into8groups:normal blood sugar control (NMC) group, diabetes control (DMC) group, diabetes ischemic (DMI) group, Yiqihuoxue (YQHX) group, local injection BMSCs (L-BMSCs) group, L-BMSCs+YQHX group, acupoint injection BMSCS (Acu-BMSCs) group, and Acu-BMSCs+YQHX group,10rats per group. Establishing animal model of diabetic mellitus:50mg/kg of Streptozotocin (STZ) per rat was injected intraperitoneally. Ischemic model:arteria femoralis as well as all of collateral artery in the left hind limb of the rats were excised. The general state, level of blood sugar and weight were observed at the2nd,7th,14th and21th day after modeling. The change of blood flow in hind limb of the rats was detected by Laser Doppler Perfusion Imager (PeriScan PIM). Immunohisto-chemistry was used to detect the vascular density which signed by a-actin, platelet endothelial cell adhesion molecule-1(PECAM-1/CD31), and von Willebrand factor (vWF), and the expression of vascular endothelial cell growth factor (VEGF) and basic fibroblast growth factor (bFGF) in muscular tissue. Elisa method was used to detect the serum level of VEGF, G-CSF and SDF-1.
Results:Most of the BMSCs obtained by all cells adherent culture method was elliptical and long-fusiform shape, and grow spirally in vitro. The membranes of third generation of BMSCs were positive for CD44and CD106and negative for CD45and CD34detected by flow cytometry. BMSCs cultured by various medicated serum increased significantly on the2nd,4th and5th day compared with the previous day. On the6th day, BMSCs proliferation by HX serum and YQHX serum began to decrease, on the other hand, the other three groups reached a peak proliferation, and decreased on the7th day. There was no significant difference among each group on the2nd day, but BMSCs cultured by HX serum increased the most. On the4th day, BMSCs cultured by YQHX serum was highest of all groups, and HX serum group was the second. On the6th day, BMSCs YQHX group was second then followed by HX group which was still higher than the control group.(2) Among different concentration of medicated serum, only10%YQHX serum cultured BMSCs began to increased significantly on the2nd day. BMSCs cultured by20%control and YQHX serum were higher than the10%FBS group. In vivo, there was87.14%rats induced diabetes. The DM rat model rat of DM was high and stable. The survival rate of ischemic rat model established by excising arterial femoral is as well as all of collateral artery in the left hind limb was88.52%in DM rats.(1) Ethological results:the rat models exhibited cachexia, pallid and pale limb, even turning dark purple, claudication and ulcer in some. The PUI of the DM rats was lower than the control group.
(2) The PUI of ischemic hind limb of the rats in the Acu-BMSCs+YQHX group increased most obviously, second were Acu-BMSCs and L-BMSCs+YQHX group, and lastly the YQHX and Acu-BMSCs group.(3) The expression of a-actin and CD31in the ischemic limb were higher than the healthy limb in each group. The expression of VEGF and bFGF mRNA, a-actin and CD31increased, and VWF decreased in the Acu-BMSCs+YQHX group compared with other groups. The levels of VEGF, SDF-1and G-CSF in rat serum tested by ELISA of Acu-BMSCs and Acu-BMSCs+group were higher than other groups.
Conclusion:The rat BMSCs cultured by all cells adherent culture method has features of BMSCs in cellular morphology and phenotypic expression.(1) The influence of HX serum on proliferation capacity of BMSCs appeared earliest of all. The influence of YQ serum on proliferation activation of BMSCs continued longest of all. The influence of YQHX serum on proliferous capacity of BMSCs was biggest of all. In vitro, the proliferous capacity of BMSCs was markedly increased obviously by10%concentration of YQHXsemn.(2) The blood flow of the ischemic hind limb of the DM rats improved efficiency and the expression of VEGF and bFGF increased by injecting BMSCs and YQHX medicine at acupuncture points. It also enhances the hyperplasia of vascular endothelial cells and vascular smooth muscle cells and prevents atherosclerosis.(3)The expression of VEGF, SDF-1and G-SGF in serum was increased in injection BMSCs group and Acu-BMSCs+YQHX medicine group.
引文
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