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[1]楊曉強,肖歡,田佳慶,等.活血通絡(luò)膠囊對激素性股骨頭壞死囊性變成血管修復的影響[J].中醫(yī)正骨,2024,36(09):40-48.
　YANG Xiaoqiang,XIAO Huan,TIAN Jiaqing,et al.Effects of Huoxue Tongluo Jiaonang(活血通絡(luò)膠囊)on angiogenesis repair of cystic lesions of steroid-induced osteonecrosis of the femoral head[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2024,36(09):40-48.
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活血通絡(luò)膠囊對激素性股骨頭壞死囊性變成血管修復的影響()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第36卷
期數(shù):
2024年09期
頁碼:
40-48
欄目:
技術(shù)研究
出版日期:
2024-09-20

文章信息/Info

Title:
Effects of Huoxue Tongluo Jiaonang(活血通絡(luò)膠囊)on angiogenesis repair of cystic lesions of steroid-induced osteonecrosis of the femoral head
作者:
楊曉強1; 肖歡2; 田佳慶1; 方偉華1; 彭鵬1; 肖方駿1; 何偉3; 何敏聰3; 魏秋實3
(1.廣州中醫(yī)藥大學第三臨床醫(yī)學院,廣東 廣州 510006; 2.畢節(jié)市中醫(yī)醫(yī)院,貴州 畢節(jié) 551700; 3.廣州中醫(yī)藥大學第三附屬醫(yī)院,廣東 廣州 510378)
Author(s):
YANG Xiaoqiang1; XIAO Huan2; TIAN Jiaqing1; FANG Weihua1; PENG Peng1; XIAO Fangjun1; HE Wei3; HE Mincong3; WEI Qiushi3
1.The Third Clinical Medical College of Guangzhou University of Chinese Medicine,Guangzhou 510006,Guangdong,China 2.Bijie Traditional Chinese Medicine Hospital,Bijie 551700,Guizhou,China 3.The Third Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510378,Guangdong,China
關(guān)鍵詞:
股骨頭壞死;  囊性變;  成血管細胞;  人臍靜脈內(nèi)皮細胞;  活血通絡(luò);  成血管修復
Keywords:
femur head necrosis;  cystic lesions;  hemangioblasts;  human umbilical vein endothelial cells;  activating blood dredging collaterals;  angiogenesis repair
摘要:
目的:探討活血通絡(luò)膠囊對激素性股骨頭壞死(steroid-induced osteonecrosis of the femoral head,SONFH)囊性變成血管修復的影響。方法:①將從全髖關(guān)節(jié)置換術(shù)中收集的一部分SONFH囊性變組織制備成單細胞懸液,采用單細胞轉(zhuǎn)錄組學分析囊性變組織中的細胞組分。將另一部分SONFH囊性變組織制成病理切片,采用蘇木素-伊紅染色法觀察囊性變中血管形態(tài),并采用免疫熒光法觀察囊性變中血管內(nèi)皮細胞特異性蛋白的表達情況。②分別按照每天20 mg·kg-1、40 mg·kg-1、80 mg·kg-1體質(zhì)量以濃度1 mg·mL-1、2 mg·mL-1、4 mg·mL-1的活血通絡(luò)膠囊溶液和20 mL·kg-1生理鹽水給大鼠灌胃,獲取低、中、高劑量活血通絡(luò)膠囊含藥血清及空白血清。③取生長良好的人臍靜脈內(nèi)皮細胞(human umbilical vein endothelial cells,HUVECs),分為空白血清組和低、中、高劑量含藥血清組,空白血清組細胞常規(guī)培養(yǎng),低、中、高劑量含藥血清組細胞分別采用低、中、高劑量活血通絡(luò)膠囊含藥血清干預。干預48 h后,通過細胞計數(shù)試劑盒-8法測定活血通絡(luò)膠囊含藥血清對HUVECs增殖能力的影響。④將HUVECs 分為對照組、地塞米松組和活血通絡(luò)組,對照組細胞常規(guī)培養(yǎng),地塞米松組細胞采用地塞米松干預,活血通絡(luò)組細胞采用地塞米松和中劑量活血通絡(luò)膠囊含藥血清干預。干預至細胞長滿后進行細胞劃痕實驗,觀察活血通絡(luò)膠囊含藥血清對HUVECs遷移能力的影響。⑤HUVECs分組與干預方法同④,干預7 d后進行成血管實驗,觀察活血通絡(luò)膠囊含藥血清對HUVECs成血管能力的影響。結(jié)果:①SONFH囊性變組織中細胞組分分析結(jié)果。SONFH囊性變組織中存在內(nèi)皮細胞及具有血管生成功能的內(nèi)皮細胞亞群。②SONFH囊性變組織病理學觀察結(jié)果。蘇木素-伊紅染色結(jié)果顯示,SONFH囊性變組織中存在橢圓形的血管腔,血管內(nèi)膜稍增厚、彈力纖維增生分層。免疫熒光染色結(jié)果顯示,SONFH囊性變組織內(nèi)存在血管內(nèi)皮細胞特異性蛋白(CD31蛋白和血管內(nèi)皮生長因子蛋白),且可見明顯的管形。③HUVECs相對增殖率測定結(jié)果。各組HUVECs相對增殖率比較,差異有統(tǒng)計學意義[0%,(0.15±0.83)%,(18.70±6.10)%,(24.9±15.10)%,F=7.541,P=0.001]; 低劑量含藥血清組HUVECs相對增殖率與空白血清組的差異無統(tǒng)計學意義(P=0.999),中劑量含藥血清組和高劑量含藥血清組HUVECs相對增殖率均高于空白血清組(P=0.046,P=0.006),中劑量含藥血清組HUVECs相對增殖率與高劑量含藥血清組的差異無統(tǒng)計學意義(P=0.782)。④HUVECs遷移率測算結(jié)果。地塞米松組HUVECs遷移率(10%)明顯低于對照組(26%),活血通絡(luò)組HUVECs遷移率(24%)明顯高于地塞米松組(10%)。⑤HUVECs形成的管形分支數(shù)測算結(jié)果。每個放大40倍視野下,各組HUVECs形成的管形分支數(shù)比較,差異有統(tǒng)計學意義[(112.20±7.96)個,(83.40±14.02)個,(122.40±14.59)個,F=2.980,P=0.001]; 地塞米松組HUVECs形成的管形分支數(shù)少于對照組和活血通絡(luò)組(P=0.006,P=0.001),對照組HUVECs形成的管形分支數(shù)與活血通絡(luò)組的差異無統(tǒng)計學意義(P=0.292)。結(jié)論:活血通絡(luò)膠囊可通過提高血管內(nèi)皮細胞的增殖、遷移和成血管能力,促進SONFH囊性變的成血管修復。
Abstract:
Objective:To explore the effects of Huoxue Tongluo Jiaonang(活血通絡(luò)膠囊,HXTLJN)on angiogenesis repair of cystic lesions of steroid-induced osteonecrosis of femoral head(SONFH).Methods:①The tissues collected from the site of cystic lesions during the total hip arthroplasty in patients with SONFH were acquired,one part was made into single-cell suspension to analyze the cellular components in cystic lesions tissues based on the single cell transcriptomics,while,the other part was made into pathological sections to observe the vascular morphology and detect the expression of vascular endothelial cells(VECs)-specific proteins in cystic lesions tissues by using hematoxylin-eosin(HE)staining and immunofluorescence method,respectively.②Twelve rats were selected and randomized into 4 groups.The rats in any three groups were intragastric administrated with HXTLJN solution at concentration of 1,2 and 4 mg/mL in daily dosage of 20,40 and 80 mg/kg,respectively,while,the ones in the last group with normal saline in daily dosage of 20 mL/kg,for making low-,medium-,and high-dose HXTLJN medicated serum and blank serum,respectively.③The well-growned human umbilical vein endothelial cells(HUVECs)were fetched and divided into blank serum group,and low-,medium-,and high-dose medicated serum groups.The HUVECs in blank serum group were cultured in the conventional Dulbecco's Modified Eagle's Medium(DMEM),while the ones in low-,medium-,and high-dose medicated serum groups were intervened with low-,medium-,and high-dose HXTLJN medicated serum,respectively.After 48-hour intervention,the effect of HXTLJN medicated serum on the proliferation ability of HUVECs was determined by using the cell counting kit-8(CCK8)assay.④The HUVECs were further divided into control group,dexamethasone(DEX)group and Huoxue Tongluo(活血通絡(luò),HXTL)group.The HUVECs in control group were cultured in the conventional DMEM,while the ones in DEX group were intervened with DEX,and the ones in HXTL group with DEX and medium-dose HXTLJN medicated serum.After the cells reached 100% confluence,the effect of HXTLJN medicated serum on the migration ability of HUVECs was observed by employing a cell scratch assay.⑤The grouping and intervention methods for HUVECs were the same as those in item ④.After 7-day intervention,the effect of HXTLJN medicated serum on the angiogenesis ability of HUVECs was observed by a tube formation assay.Results:①The results of analysis on the cellular components in the SONFH-associated cystic lesions tissues.The endothelial cells and endothelial cell subsets with angiogenesis function existed in the SONFH-associated cystic lesions tissues.②The results of observation on histopathological changes in SONFH-associated cystic lesions tissues.The results of HE staining showed that the oval-shaped vascular lumens,with slightly thickened vascular intima and hyperplastic,layered elastic fibers,were presented in the SONFH-associated cystic lesions tissues.The results of immunofluorescence staining revealed that the VEC-specific proteins(CD31 and vascular endothelial growth factor)existed in the SONFH-associated cystic lesions tissues,and the obvious tubular structures were observed.③The results of detection on the relative proliferation rate of HUVECs.The difference was statistically significant among the groups in the relative proliferation rate of HUVECs(0%,0.15±0.83,18.70±6.10,24.9±15.10%,F=7.541,P=0.001).The relative proliferation rate of HUVECs were higher in medium- and high-dose medicated serum groups compared to blank serum group(P=0.046,P=0.006),while,the comparisons between low-dose medicated serum group and blank serum group,as well as between medium-dose medicated serum group and high-dose medicated serum group revealed no significant differences(P=0.999; P=0.782).④The results of calculation on migration rate of HUVECs.The migration rate of HUVECs was significantly lower in DEX group(10%)compared to control group(26%),while,it was obviously higher in HXTL group(24%)compared to DEX group(10%).⑤The results of calculation on the number of tube-like branches formed by HUVECs.Under the 40-fold magnification fields,the difference was statistically significant among the groups in the number of tube-like branches formed by HUVECs(112.20±7.96,83.40±14.02,122.40±14.59 branches,F=2.980,P=0.001).The tube-like branches formed by HUVECs were less in DEX group compared to control group and HXTL group(P=0.006,P=0.001),while,the comparison between control group and HXTL group revealed no significant difference(P=0.292).Conclusion:The HXTLJN can promote the angiogenesis repair in SONFH-associated cystic lesions by enhancing the proliferation,migration,and angiogenesis capabilities of VECs.

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備注/Memo

備注/Memo:
基金項目:國家自然科學基金項目(8227150353,82004392); 廣東省基礎(chǔ)與應用基礎(chǔ)研究基金項目(2023A1515010551); 廣東省中醫(yī)骨傷研究院開放課題重點項目(GYH202101-01,GYH202101-04); 畢節(jié)市科學技術(shù)局2022年度“揭榜掛帥”項目(畢科合重大專項〔2022〕1號); 中國博士后科學基金第75批面上資助項目(2024M750819); 中醫(yī)證候全國重點實驗室研究生項目(SKLKY2024A0003)
通訊作者:魏秋實 E-mail:[email protected]
更新日期/Last Update: 1900-01-01