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[1]彭鵬,肖歡,方偉華,等.激素性股骨頭壞死囊性變組織骨修復(fù)機制分析與活血通絡(luò)膠囊含藥血清對肥大軟骨細胞成骨分化影響的實驗研究[J].中醫(yī)正骨,2024,36(09):19-28.
　PENG Peng,XIAO Huan,FANG Weihua,et al.[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2024,36(09):19-28.
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激素性股骨頭壞死囊性變組織骨修復(fù)機制分析與活血通絡(luò)膠囊含藥血清對肥大軟骨細胞成骨分化影響的實驗研究()
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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:
19-28
欄目:
技術(shù)研究
出版日期:
2024-09-20

文章信息/Info

作者:
彭鵬1; 肖歡2; 方偉華1; 田佳慶1; 林錕1; 姚放鳴1; 肖方駿1; 何敏聰3; 何偉3; 魏秋實3
(1.廣州中醫(yī)藥大學(xué)第三臨床醫(yī)學(xué)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(xué)第三附屬醫(yī)院,廣東 廣州 510378)
Author(s):
PENG Peng1; XIAO Huan2; FANG Weihua1; TIAN Jiaqing1; LIN Kun1; YAO Fangming1; XIAO Fangjun1; HE Mincong3; HE Wei3; 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;  糖皮質(zhì)激素;  囊性變;  RNA測序;  活血通絡(luò)膠囊;  軟骨細胞;  細胞分化;  骨生成
Keywords:
femur head necrosis;  glucocorticoids;  cystic lesions;  RNA-Seq;  Huoxue Tongluo capsule;  chondrocytes;  cell differentiation;  osteogenesis
摘要:
目的:分析激素性股骨頭壞死(steroid-induced osteonecrosis of the femoral head,SONFH)囊性變組織的骨修復(fù)機制,并觀察活血通絡(luò)膠囊含藥血清對肥大軟骨細胞成骨分化的影響。方法:①SONFH囊性變組織的骨修復(fù)機制分析。納入6例采用全髖關(guān)節(jié)置換術(shù)治療的SONFH患者,術(shù)后收集壞死股骨頭6個,其中3個股骨頭用于囊性變組織的單細胞轉(zhuǎn)錄組測序,采用生物信息學(xué)方法分析囊性變組織中的細胞分類、軟骨細胞的細胞亞群、肥大軟骨細胞參與的生物過程; 另外3個股骨頭用于囊性變組織的病理學(xué)檢查,分別采用蘇木素-伊紅染色、阿利新藍染色、番紅O-固綠染色觀察囊性變組織中軟骨細胞、肥大軟骨細胞的分布與特征,采用免疫組織化學(xué)染色觀察囊性變組織中肥大軟骨細胞標(biāo)志蛋白Ⅹ型膠原α1(collagen typeⅩα1,Col10α1)、基質(zhì)金屬蛋白酶(matrix metalloproteinase,MMP)13和成骨分化相關(guān)蛋白Runt相關(guān)轉(zhuǎn)錄因子2(Runt-related transcription factor 2,RUNX2)、骨橋蛋白(osteopontin,OPN)的表達情況。②活血通絡(luò)膠囊含藥血清對肥大軟骨細胞成骨分化影響的分析。采用活血通絡(luò)膠囊(活血通絡(luò)膠囊粉末溶于蒸餾水)給大鼠灌胃,制備活血通絡(luò)膠囊含藥血清。取小鼠膝關(guān)節(jié)軟骨,消化分離后進行培養(yǎng)。將小鼠軟骨細胞接種于含有不同濃度的活血通絡(luò)膠囊含藥血清的完全培養(yǎng)基中,篩選活血通絡(luò)膠囊含藥血清的最佳干預(yù)濃度。取處于對數(shù)生長期的第1代小鼠軟骨細胞,行肥大軟骨細胞誘導(dǎo)成功后,分為成骨誘導(dǎo)組、5%含藥血清干預(yù)組和對照組,成骨誘導(dǎo)組更換成骨誘導(dǎo)培養(yǎng)基培養(yǎng),5%含藥血清干預(yù)組更換含5%活血通絡(luò)膠囊含藥血清的成骨誘導(dǎo)培養(yǎng)基培養(yǎng),對照組繼續(xù)采用肥大誘導(dǎo)培養(yǎng)基培養(yǎng); 培養(yǎng)7 d后,分別采用堿性磷酸酶(alkaline phosphatase,ALP)染色和茜素紅染色,觀察細胞形態(tài)特征,分別采用實時定量PCR和蛋白質(zhì)印跡法檢測Ⅰ型膠原蛋白α1(collagen Ⅰα1,Col1α1)、ALP、RUNX2、OPN的mRNA相對表達量和ALP、RUNX2、OPN的蛋白相對表達量。結(jié)果:①SONFH囊性變組織單細胞轉(zhuǎn)錄組測序分析結(jié)果。共獲得30 224個細胞的單細胞轉(zhuǎn)錄組測序結(jié)果; 細胞分類結(jié)果顯示,囊性變組織中有8種細胞,包括T細胞、內(nèi)皮細胞、成纖維細胞、軟骨細胞、巨噬細胞、單核細胞、破骨細胞和肥大細胞; 軟骨細胞亞群分析共鑒定出5類亞群細胞,包括穩(wěn)態(tài)軟骨細胞、成纖維軟骨細胞、炎癥軟骨細胞、肥大軟骨細胞、前體肥大軟骨細胞; 差異基因富集分析結(jié)果顯示,肥大軟骨細胞主要參與細胞遷移正向調(diào)節(jié)、細胞分化、細胞外基質(zhì)形成、骨化、細胞遷移和成骨細胞分化等生物過程。②SONFH囊性變組織的病理學(xué)檢查結(jié)果。囊性變組織的邊緣可見軟骨細胞、肥大軟骨細胞,且部分軟骨組織呈現(xiàn)向骨小梁過渡的形態(tài); Col10α1、MMP13、RUNX2、OPN在囊性變組織的邊緣高表達。③活血通絡(luò)膠囊含藥血清干預(yù)肥大軟骨細胞成骨分化的分析結(jié)果。經(jīng)篩選,5%為活血通絡(luò)膠囊含藥血清的最佳干預(yù)濃度。ALP染色和茜素紅染色結(jié)果顯示,對照組無明顯鈣化結(jié)節(jié),成骨誘導(dǎo)組有明顯鈣化結(jié)節(jié),5%含藥血清干預(yù)組鈣化結(jié)節(jié)較成骨誘導(dǎo)組進一步增多; 成骨誘導(dǎo)組細胞ALP、RUNX2、OPN、Col1α1的mRNA相對表達量和ALP、RUNX2、OPN的蛋白相對表達量均高于對照組(P=0.000,P=0.000,P=0.001,P=0.000; P=0.000,P=0.000,P=0.001),5%含藥血清干預(yù)組細胞ALP、RUNX2、OPN、Col1α1的mRNA相對表達量和ALP、RUNX2、OPN的蛋白相對表達量均高于成骨誘導(dǎo)組(P=0.000,P=0.000,P=0.002,P=0.001; P=0.000,P=0.000,P=0.001)。結(jié)論:SONFH囊性變組織中存在肥大軟骨細胞向成骨細胞分化的修復(fù)機制,而活血通絡(luò)膠囊含藥血清能夠促進體外培養(yǎng)的肥大軟骨細胞向成骨細胞分化。
Abstract:
Objective:To analyze the mechanism of bone repair in cystic lesion tissues of steroid-induced osteonecrosis of the femoral head(SONFH),and to observe the effects of Huoxue Tongluo Jiaonang(活血通絡(luò)膠囊,HXTLJN)medicated serum on the osteogenic differentiation of hypertrophic chondrocytes(HCs).Methods:①Analyzing the mechanism of bone repair in SONFH-associated cystic lesion tissues.Six patients who underwent total hip arthroplasty for SONFH were enrolled in the study,and their necrotic femur heads were collected after the surgery.Among the 6 femur heads,3 ones were used for single-cell RNA sequencing(scRNA-seq)on cystic lesion tissues to analyze the cell categories,chondrocyte subsets,and biological processes involving in HCs in cystic lesion tissues by bioinformatics methods; while,the other 3 ones were used for pathological examination on the cystic lesion tissues to observe the distributions and characteristics of chondrocytes and HCs in cystic lesion tissues by using hematoxylin-eosin(HE)staining,Alcian blue staining,and safranin O-fast green(SO-FG)staining,respectively,and detect the expression of hypertrophic chondrocyte marker protein including collagen typeⅩα1(Col10α1)and matrix metalloproteinase(MMP)13,as well as the osteogenic differentiation-related protein including Runt-related transcription factor 2(RUNX2)and osteopontin(OPN)in the cystic lesion tissues by using immunohistochemical staining.②Analyzing the effects of HXTLJN medicated serum on the osteogenic differentiation of HCs.The rats were intervened by intragastric administration with HXTLJN(HXTLJN powders were dissolved into the distilled water)for making HXTLJN medicated serum.The knee articular cartilages were harvested from the sacrificed mice,and were digested and separated for chondrocytes,which were then cultured in DMEM/F-12.Furthermore,the chondrocytes were inoculated into complete medium containing different concentrations of HXTLJN medicated serum for determining the optimal intervention concentration of HXTLJN medicated serum.The HCs were induced from the first-generation mouse chondrocytes in the logarithmic phase,after successful induction,they were divided into osteogenic induction group,5% HXTLJN medicated serum intervention group,and control group.The HCs in osteogenic induction group were cultured with osteogenesis inducing medium(OIM),the ones in 5% HXTLJN medicated serum intervention group with OIM containing 5% HXTLJN medicated serum,while the ones in control group with hypertrophy-inducing medium.After 7-day culture,the morphological characteristics of the HCs were observed by using alkaline phosphatase(ALP)staining and alizarin red(AR)staining,respectively; and the relative mRNA expression levels of collagen Iα1(Col1α1),ALP,RUNX2 and OPN,as well as the relative protein expression levels of ALP,RUNX2 and OPN were detected by using real-time quantitative PCR(RT-qPCR)and Western blotting,respectively.Results:①The results of analysis on SONFH-associated cystic lesion tissues by scRNA-seq.The scRNA-seq results of 30 224 cells were acquired.The results of cell categories indicated that the cystic lesions harbored eight categories of cells,including T cells,endothelial cells,fibroblasts,chondrocytes,macrophages,monocytes,osteoclasts,and mast cells.The results of chondrocyte subset analysis showed that the chondrocytes included five subsets,namely homeostatic chondrocytes,fibroblast chondrocytes,inflammatory chondrocytes,hypertrophic chondrocytes,and precursor hypertrophic chondrocytes.The results of enrichment analysis on the differentially expressed genes(DEGs)showed that the hypertrophic chondrocytes mainly participated in the biological processes such as positive regulation of cell migration,cell differentiation,extracellular matrix formation,ossification,cell migration,and osteoblast differentiation.②The results of examination on histopathological changes in SONFH-associated cystic lesion tissues.The cartilage cells and HCs were found at the edge of cystic lesion tissues,with some cartilage tissue presented a transition towards to bone trabeculae in morphology.Moreover,the Col10α1,MMP13,RUNX2,and OPN were highly expressed at the edge of cystic lesion tissues.③The results of analysis on the effects of HXTLJN medicated serum in interventing osteogenic differentiation of HCs.The screening results showed the optimal intervention concentration of HXTLJN medicated serum was 5%.The ALP and ARS staining results showed that the distinct calcified nodules were found in osteogenic induction group,which,however,failed to be found in control group,and they were further increased in 5% HXTLJN medicated serum intervention group compared to osteogenic induction group.Additionally,the relative mRNA expression levels of ALP,RUNX2,OPN and Col1α1,as well as the relative protein expressions levels of ALP,RUNX2 and OPN were higher in osteogenic induction group compared to control group(P=0.000,P=0.000,P=0.001,P=0.000; P=0.000,P=0.000,P=0.001),and were highest in 5% HXTLJN medicated serum intervention group(P=0.000,P=0.000,P=0.002,P=0.001; P=0.000,P=0.000,P=0.001).Conclusion:A repair mechanism of hypertrophic chondrocytes differentiating into osteoblasts is presented in the cystic lesion tissues of SONFH,which can be promoted by the HXTLJN medicated serum in vitro.

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備注/Memo

備注/Memo:
基金項目:國家自然科學(xué)基金項目(82274544,82004392); 廣東省基礎(chǔ)與應(yīng)用基礎(chǔ)研究基金項目(2023A1515010551); 畢節(jié)市科學(xué)技術(shù)局2022年度“揭榜掛帥”項目(畢科合重大專項〔2022〕1號); 廣東省中醫(yī)骨傷研究院開放課題重點項目(GYH202101-01,GYH202101-04) 通訊作者:魏秋實 E-mail:[email protected]
更新日期/Last Update: 1900-01-01