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Article(id=1200026649539478353, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1200026645001241395, articleNumber=null, orderNo=null, doi=10.11855/j.issn.0577-7402.2338.2023.0601, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1667923200000, receivedDateStr=2022-11-09, revisedDate=null, revisedDateStr=null, acceptedDate=1673193600000, acceptedDateStr=2023-01-09, onlineDate=1764038248667, onlineDateStr=2025-11-25, pubDate=1693152000000, pubDateStr=2023-08-28, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1764038248667, onlineIssueDateStr=2025-11-25, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1764038248667, creator=13701087609, updateTime=1764038248667, updator=13701087609, issue=Issue{id=1200026645001241395, tenantId=1146029695717560320, journalId=1189873630562394117, year='2023', volume='48', issue='8', pageStart='871', pageEnd='992', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1764038247584, creator=13701087609, updateTime=1764038741950, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1200028718564474883, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1200026645001241395, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1200028718564474884, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1200026645001241395, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=879, endPage=886, ext={EN=ArticleExt(id=1200026649916965719, articleId=1200026649539478353, tenantId=1146029695717560320, journalId=1189873630562394117, language=EN, title=Effect of stem cells from human exfoliated deciduous teeth on temporomandibular joint osteoarthritis in rats, columnId=1190310110212751762, journalTitle=Medical Journal of Chinese People’s Liberation Army, columnName=Basic Research, runingTitle=null, highlight=null, articleAbstract=

Objective To investigate the effects of stem cells from human deciduous teeth (SHED) on temporomandibular joint osteoarthritis (TMJOA) in rats. Methods Sixty 8-week-old male SD rats were randomly divided into control group, sodium iodoacetate (MIA)-induced TMJOA group (MIA group) and SHED treatment TMJOA group (SHED group), 20 rats in each group, and 10 animals were sacrificed in each group 2 and 4 weeks after treatment and were collected. Temporomandibular joint (TMJ) on the left was used for morphological detection, and right TMJ condylar cartilage for molecular biology detection. The degree of cartilage degeneration was evaluated by HE and Saffron O-solid green staining, the expression of type collagen Ⅱ in condylar cartilage was detected by immunohistochemical staining, and the expression changes of cleaved-CASP3, the key molecule of apoptosis, and the pro-inflammatory factor tumor necrosis factor-α (TNF-α), were detected by Western blotting. Results Compared with control group, the arrangement of cells in each layer of condylar cartilage in MIA group was disordered, a large number of cell-free areas were visible, and the fibrous layer was significantly thickened (P<0.001). After SHED treatment, the morphology of SHED group basically returned to normal, and there was no significant difference between SHED group and control group. The histological score of Mankin's osteoarthritis of condylar cartilage in MIA group was significantly higher than that in control group (P<0.001). After treatment, the score decreased significantly in SHED group (P<0.001). The positive area ratio of Saffron-O staining and the percentage of positive area of collagen Ⅱ. Of condylar cartilage in MIA group were significantly lower than those in control group (P<0.001). After treatment, the value increased significantly in SHED group (P<0.01) and there was no significant difference compared with control group. The number of TUNEL-positive cells in the condylar cartilage in MIA group was significantly higher than that in control group (P<0.001). After treatment, this value decreased significantly in SHED group (P<0.001). Western blotting results showed that the protein expression levels of cleaved-CASP3 and TNF-α in the condylar cartilage of MIA group were significantly higher than those in control group (P<0.001). After treatment, this value decreased significantly in SHED group (P<0.001) and there was no significant difference compared with control group. Conclusion Intra-articular injection of SHED can reverse condylar cartilage degeneration induced by MIA.

, correspAuthors=Shi-Bin Yu, Jian-Liang Pang, authorNote=null, correspAuthorsNote=
Pang Jian-Liang, E-mail:
Yu Shi-Bin, E-mail:
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目的 探讨人脱落乳牙牙髓间充质干细胞(SHED)对大鼠颞下颌关节骨关节炎(TMJOA)的治疗作用。方法 60只8周龄雄性SD大鼠随机分为对照组、碘乙酸钠(MIA)诱导致TMJOA组(MIA组)、SHED治疗TMJOA组(SHED组)，每组20只，各组分别于处理后2、4周处死10只并取材，左侧颞下颌关节(TMJ)用于形态学检测，右侧TMJ髁突软骨用于分子生物学检测。采用HE、番红O-固绿染色评价软骨退变程度，免疫组化染色检测髁突软骨中Ⅱ型胶原的表达情况，Western blotting检测凋亡关键分子cleaved-CASP3、促炎因子肿瘤坏死因子-α(TNF-α)等的表达变化。结果 与对照组相比，MIA组髁突软骨各层细胞排列紊乱，可见大量无细胞区，且纤维层明显增厚(P<0.001)；经SHED治疗后，SHED组形态基本恢复正常，与对照组比较无明显差异。MIA组髁突软骨的骨关节炎改良Mankin组织学评分明显高于对照组(P<0.001)，SHED组经治疗后明显降低(P<0.001)，且与对照组比较差异无统计学意义。MIA组髁突软骨的番红O染色阳性面积比和Ⅱ型胶原阳性面积百分比均明显低于对照组(P<0.001)，SHED组经治疗后较MIA组明显增高(P<0.01)，且与对照组比较差异无统计学意义。MIA组髁突软骨中的TUNEL阳性细胞数明显多于对照组(P<0.001)，SHED组经治疗后较MIA组明显减少(P<0.001)，但与对照组比较差异仍有统计学意义(2周，P<0.01；4周，P<0.001)。Western blotting检测显示，MIA组髁突软骨中cleaved-CASP3和TNF-α表达水平明显高于对照组(P<0.001)，SHED组经治疗后明显降低(P<0.001)，且与对照组比较差异无统计学意义。结论 SHED治疗可以有效逆转MIA所致的大鼠髁突软骨退变。

, correspAuthors=于世宾, 逄键梁, authorNote=null, correspAuthorsNote=
逄键梁，E-mail：
于世宾，E-mail：
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何睿，硕士研究生，主要从事颞下颌关节骨关节炎修复方面的研究

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何睿，硕士研究生，主要从事颞下颌关节骨关节炎修复方面的研究

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何睿，硕士研究生，主要从事颞下颌关节骨关节炎修复方面的研究

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TMJ. 颞下颌关节；MIA. 碘乙酸钠；SHED. 人脱落乳牙髓间充质干细胞；A. TMJ髁突矢状面HE染色；B. 软骨纤维层厚度及改良Mankin组织学评分(n=10)；箭头示软骨内无细胞区；***P<0.001

, figureFileSmall=ZIthYL6e0vmbk7VBfNS84Q==, figureFileBig=uuzLIGIRGgsREpRU4URorQ==, tableContent=null), ArticleFig(id=1200026658624340186, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1200026649539478353, language=EN, label=Fig.2, caption=Safranin O-fast green staining results of rat condylar cartilage, figureFileSmall=yh/56XWEAoCCZ52tNwTeoQ==, figureFileBig=iLSrBOG1lUp9JAH06tgacw==, tableContent=null), ArticleFig(id=1200026658725003489, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1200026649539478353, language=CN, label=图2, caption=大鼠髁突软骨番红O-固绿染色结果

TMJ. 颞下颌关节；MIA. 碘乙酸钠；SHED. 人脱落乳牙髓间充质干细胞；A. 大鼠TMJ髁突矢状面番红O-固绿染色；B. 番红O染色阳性面积统计图(n=10)；箭头指示软骨内无细胞区；**P<0.01，***P<0.001

, figureFileSmall=yh/56XWEAoCCZ52tNwTeoQ==, figureFileBig=iLSrBOG1lUp9JAH06tgacw==, tableContent=null), ArticleFig(id=1200026658821472486, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1200026649539478353, language=EN, label=Fig.3, caption=Immunohistochemical staining results of collagen Ⅱ in rat condylar cartilage (SP), figureFileSmall=L0IanzfxE0caG1AvO3WpeA==, figureFileBig=jmPGicpp6zU4J01pA3uV8Q==, tableContent=null), ArticleFig(id=1200026658951495915, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1200026649539478353, language=CN, label=图3, caption=大鼠髁突软骨Ⅱ型胶原免疫组化染色结果(SP法)

TMJ. 颞下颌关节；MIA. 碘乙酸钠；SHED. 人脱落乳牙髓间充质干细胞；A. TMJ髁突矢状面Ⅱ型胶原免疫组化染色；B. Ⅱ型胶原阳性面积统计图(n=10)；箭头指示软骨内无细胞区；**P<0.01，***P<0.001

, figureFileSmall=L0IanzfxE0caG1AvO3WpeA==, figureFileBig=jmPGicpp6zU4J01pA3uV8Q==, tableContent=null), ArticleFig(id=1200026659052159217, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1200026649539478353, language=EN, label=Fig.4, caption=TUNEL staining results in rat condylar cartilage, figureFileSmall=KhMW3DfL9Y52uS8WZrPW6w==, figureFileBig=VmoFx9L7zEp4e2MdwkYq+Q==, tableContent=null), ArticleFig(id=1200026659169599734, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1200026649539478353, language=CN, label=图4, caption=大鼠髁突软骨TUNEL染色结果

TMJ. 颞下颌关节；MIA. 碘乙酸钠；SHED. 人脱落乳牙髓间充质干细胞；A. TMJ髁突矢状面TUNEL染色；B. TUNEL阳性细胞率统计图(n=10)；**P<0.01，***P<0.001

, figureFileSmall=KhMW3DfL9Y52uS8WZrPW6w==, figureFileBig=VmoFx9L7zEp4e2MdwkYq+Q==, tableContent=null), ArticleFig(id=1200026659295428863, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1200026649539478353, language=EN, label=Fig.5, caption=Changes of protein expression of cleaved-CASP3 and TNF-α in rat condylar cartilage (n=3), figureFileSmall=ew3kvxvVyWQJUEms5RojRg==, figureFileBig=xsWTobWnpMNd2ksZd3ITBQ==, tableContent=null), ArticleFig(id=1200026659417063682, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1200026649539478353, language=CN, label=图5, caption=各组大鼠髁突软骨中cleaved-CASP3、TNF-α蛋白的表达变化(n=3)

MIA. 碘乙酸钠；SHED. 人脱落乳牙髓间充质干细胞；A. cleaved-CASP3和TNF-α蛋白表达的Western blotting检测；B. cleaved-CASP3和TNF-α蛋白相对表达水平的柱状统计图；**P<0.01，***P<0.001

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人脱落乳牙牙髓间充质干细胞对大鼠颞下颌关节骨关节炎的治疗作用
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何睿 1 , 陈晓华 2 , 何峰 2 , 马原军 2 , 吴凡 2 , 段宇辰 1 , 苗辉 2 , 于世宾 2, * , 逄键梁 1, *
解放军医学杂志 | 基础研究 2023,48(8): 879-886
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解放军医学杂志 | 基础研究 2023, 48(8): 879-886
人脱落乳牙牙髓间充质干细胞对大鼠颞下颌关节骨关节炎的治疗作用
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何睿1, 陈晓华2, 何峰2, 马原军2, 吴凡2, 段宇辰1, 苗辉2, 于世宾2, * , 逄键梁1, *
作者信息
  • 1安徽医科大学空军临床学院/安徽医科大学第五临床医学院/空军特色医学中心口腔科，北京 100142
  • 2空军军医大学口腔医院解剖生理学教研室/军事口腔医学国家重点实验室/口腔疾病国家临床医学研究中心/陕西省口腔疾病国际联合研究中心，陕西西安 710032

    • 何睿，硕士研究生，主要从事颞下颌关节骨关节炎修复方面的研究

通讯作者:

逄键梁，E-mail：
于世宾，E-mail：
Effect of stem cells from human exfoliated deciduous teeth on temporomandibular joint osteoarthritis in rats
Rui He1, Xiao-Hua Chen2, Feng He2, Yuan-Jun Ma2, Fan Wu2, Yu-Chen Duan1, Hui Miao2, Shi-Bin Yu2, * , Jian-Liang Pang1, *
Affiliations
  • 1Air Force Clinical Medical College of Anhui Medical University/the Fifth Clinical Medical College of Anhui Medical University/Department of Stomatology, Air Force Specialty Medical Center of Chinese PLA, Beijing 100142, China
  • 2Department of Anatomy and Physiology, Stomatological Hospital, Air Force Military Medical University/State Key Laboratory of Military Oral Medicine/National Clinical Medical Research Center for Oral Diseases/Shaanxi International Joint Research Center for Oral Diseases, Xi'an, Shaanxi 710032, China
出版时间: 2023-08-28 doi: 10.11855/j.issn.0577-7402.2338.2023.0601
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摘要
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目的 探讨人脱落乳牙牙髓间充质干细胞(SHED)对大鼠颞下颌关节骨关节炎(TMJOA)的治疗作用。方法 60只8周龄雄性SD大鼠随机分为对照组、碘乙酸钠(MIA)诱导致TMJOA组(MIA组)、SHED治疗TMJOA组(SHED组)，每组20只，各组分别于处理后2、4周处死10只并取材，左侧颞下颌关节(TMJ)用于形态学检测，右侧TMJ髁突软骨用于分子生物学检测。采用HE、番红O-固绿染色评价软骨退变程度，免疫组化染色检测髁突软骨中Ⅱ型胶原的表达情况，Western blotting检测凋亡关键分子cleaved-CASP3、促炎因子肿瘤坏死因子-α(TNF-α)等的表达变化。结果 与对照组相比，MIA组髁突软骨各层细胞排列紊乱，可见大量无细胞区，且纤维层明显增厚(P<0.001)；经SHED治疗后，SHED组形态基本恢复正常，与对照组比较无明显差异。MIA组髁突软骨的骨关节炎改良Mankin组织学评分明显高于对照组(P<0.001)，SHED组经治疗后明显降低(P<0.001)，且与对照组比较差异无统计学意义。MIA组髁突软骨的番红O染色阳性面积比和Ⅱ型胶原阳性面积百分比均明显低于对照组(P<0.001)，SHED组经治疗后较MIA组明显增高(P<0.01)，且与对照组比较差异无统计学意义。MIA组髁突软骨中的TUNEL阳性细胞数明显多于对照组(P<0.001)，SHED组经治疗后较MIA组明显减少(P<0.001)，但与对照组比较差异仍有统计学意义(2周，P<0.01；4周，P<0.001)。Western blotting检测显示，MIA组髁突软骨中cleaved-CASP3和TNF-α表达水平明显高于对照组(P<0.001)，SHED组经治疗后明显降低(P<0.001)，且与对照组比较差异无统计学意义。结论 SHED治疗可以有效逆转MIA所致的大鼠髁突软骨退变。

脱落乳牙牙髓间充质干细胞  /  颞下颌关节骨关节炎  /  髁突  /  软骨

Objective To investigate the effects of stem cells from human deciduous teeth (SHED) on temporomandibular joint osteoarthritis (TMJOA) in rats. Methods Sixty 8-week-old male SD rats were randomly divided into control group, sodium iodoacetate (MIA)-induced TMJOA group (MIA group) and SHED treatment TMJOA group (SHED group), 20 rats in each group, and 10 animals were sacrificed in each group 2 and 4 weeks after treatment and were collected. Temporomandibular joint (TMJ) on the left was used for morphological detection, and right TMJ condylar cartilage for molecular biology detection. The degree of cartilage degeneration was evaluated by HE and Saffron O-solid green staining, the expression of type collagen Ⅱ in condylar cartilage was detected by immunohistochemical staining, and the expression changes of cleaved-CASP3, the key molecule of apoptosis, and the pro-inflammatory factor tumor necrosis factor-α (TNF-α), were detected by Western blotting. Results Compared with control group, the arrangement of cells in each layer of condylar cartilage in MIA group was disordered, a large number of cell-free areas were visible, and the fibrous layer was significantly thickened (P<0.001). After SHED treatment, the morphology of SHED group basically returned to normal, and there was no significant difference between SHED group and control group. The histological score of Mankin's osteoarthritis of condylar cartilage in MIA group was significantly higher than that in control group (P<0.001). After treatment, the score decreased significantly in SHED group (P<0.001). The positive area ratio of Saffron-O staining and the percentage of positive area of collagen Ⅱ. Of condylar cartilage in MIA group were significantly lower than those in control group (P<0.001). After treatment, the value increased significantly in SHED group (P<0.01) and there was no significant difference compared with control group. The number of TUNEL-positive cells in the condylar cartilage in MIA group was significantly higher than that in control group (P<0.001). After treatment, this value decreased significantly in SHED group (P<0.001). Western blotting results showed that the protein expression levels of cleaved-CASP3 and TNF-α in the condylar cartilage of MIA group were significantly higher than those in control group (P<0.001). After treatment, this value decreased significantly in SHED group (P<0.001) and there was no significant difference compared with control group. Conclusion Intra-articular injection of SHED can reverse condylar cartilage degeneration induced by MIA.

mesenchymal stem cells  /  temporomandibular joint osteoarthritis  /  condyle  /  cartilage
何睿, 陈晓华, 何峰, 马原军, 吴凡, 段宇辰, 苗辉, 于世宾, 逄键梁. 人脱落乳牙牙髓间充质干细胞对大鼠颞下颌关节骨关节炎的治疗作用. 解放军医学杂志, 2023 , 48 (8) : 879 -886 . DOI: 10.11855/j.issn.0577-7402.2338.2023.0601
Rui He, Xiao-Hua Chen, Feng He, Yuan-Jun Ma, Fan Wu, Yu-Chen Duan, Hui Miao, Shi-Bin Yu, Jian-Liang Pang. Effect of stem cells from human exfoliated deciduous teeth on temporomandibular joint osteoarthritis in rats[J]. Medical Journal of Chinese People’s Liberation Army, 2023 , 48 (8) : 879 -886 . DOI: 10.11855/j.issn.0577-7402.2338.2023.0601
正文
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颞下颌关节紊乱病(temporomandibular disorders，TMD)是指累及颞下颌关节(temporomandibular join，TMJ)和(或)咀嚼肌的一类口腔疾病，其患病率高达20%~40%，主要临床表现为颌面部疼痛、关节杂音和下颌运动受限，严重影响患者的生活质量。颞下颌关节骨关节炎(temporomandibular joint osteoarthritis，TMJOA)是TMD的重症表现形式，其主要病理表现为软骨基质降解、软骨细胞死亡及软骨下骨异常改建。据统计，约14.56%的TMD患者具有TMJOA的影像学表现[1]。由于髁突软骨细胞数量有限，缺乏血管供应，软骨再生能力相对较差，因而针对TMJOA的治疗手段有限[2]。间充质干细胞(mesenchymal stem cells，MSCs)是具有自我更新和多向分化能力的多潜能前体细胞，临床研究发现其具有促进关节软骨再生、缓解症状和疼痛控制等作用，近年来已逐步应用于TMJOA的治疗[3-6]。人脱落乳牙牙髓间充质干细胞(stem cells from human exfoliated deciduous teeth，SHED)是存在于牙髓血管周围、能够自我更新的一类MSCs。与其他MSCs相比，SHED具有伦理争议少、容易获得、微创收集以及容易保留高干细胞潜能等优点[7]。大量研究发现，SHED可诱导骨和软骨形成，具有修复关节软骨、抑制炎症反应等生物学潜能，有着良好的转化应用前景[8-11]。本研究拟初步探讨SHED对TMJOA动物模型中髁突软骨退变转归的影响，以期为今后TMJOA的临床治疗提供新的思路。
1 材料与方法
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1.1 主要材料与仪器
肿瘤坏死因子-α(TNF-α)抗体(AF7014)，含半胱氨酸的天冬氨酸蛋白水解酶-3(cleaved-caspase 3，cleaved-CASP3)抗体(AF7022)，β-tubulin抗体(AF7011)(美国Affinity公司)；青链霉素双抗溶液，DMEM高糖培养基，α-MEM培养基(美国Hyclone公司)；Tripure分离试剂(美国Roche公司)；Ⅰ型、Ⅱ型胶原酶(美国Worthington公司)；碘乙酸钠(德国Sigma-Aldrich公司)；一步法TUNEL细胞凋亡检测试剂盒(上海碧云天公司)；DM2500生物显微镜(德国Leica公司)；Chemi-Doc™ XRS+WB发光成像仪(美国Bio-Rad公司)；核酸蛋白测定仪和酶标仪(德国Eppendorf公司)；聚焦激光扫描显微镜(美国Olympus公司)。
1.2 SHED的提取、培养和传代
SHED为空军军医大学口腔医院预防科轩昆教授团队赠予，提取和鉴定过程见文献[12]。脱落乳牙用PBS反复多次冲洗干净，取出牙髓组织剪碎；加入Ⅰ型胶原酶，在37 ℃孵箱内消化40 min~1 h，每隔5~10 min摇匀一次；经过消化的细胞悬液800 r/min离心5 min，弃上清，加入2 ml含10%胎牛血清和100 U/ml青霉素、链霉素的α-MEM培养液；将4×106/ml的细胞接种于75 ml培养瓶内，置于37 ℃、5% CO2恒温敷箱培养7~10 d，此代细胞标记为P0代。待细胞生长融合至80%左右时按照1∶3传代，传至P6代细胞作为后续实验所用细胞。
1.3 动物分组及模型建立
60只8周龄雄性Sprague-Dawley(SD)大鼠，体重250~280 g，购自空军军医大学实验动物中心。将大鼠随机分为对照组、碘乙酸钠(MIA)诱导TMJOA模型组(MIA组)和SHED治疗TMJOA组(SHED组)，每组20只。1%戊巴比妥钠腹腔注射深度麻醉大鼠，刮除大鼠TMJ区域的毛发，碘伏消毒。MIA组和SHED组大鼠使用微量注射器在两侧TMJ上腔内注入50 μl 4 mg/kg的MIA建立TMJOA模型，对照组则以同样方法注射等体积的PBS。SHED组在造模后7 d注射50 μl 4×106/ml SHED细胞悬液，对照组和MIA组注射等体积的PBS。各组大鼠分别于注射SHED悬液或PBS后2、4周每组各处死10只并取材，左侧TMJ用于形态学检测，右侧TMJ髁突软骨用于分子生物学检测。
1.4 HE染色
大鼠左侧TMJ固定、脱矿后，常规石蜡包埋切片，HE染色，显微镜下观察并采集图像，Photoshop软件测量软骨纤维层厚度。
1.5 番红O-固绿染色
石蜡切片常规脱蜡至水，进行番红O-固绿染色：1%固绿染色1 min，1%醋酸乙醇分化30 s，待切片自然干燥后，1%番红O染色5 min，95%乙醇漂洗15 s。显微镜下观察并采集图像，每个样本随机选取3个视野，用Photoshop软件计算番红O阳性面积百分比，观察髁突软骨基质中蛋白多糖的表达变化。
1.6 免疫组化染色
石蜡切片常规脱蜡至水，按照试剂盒说明书中描述的SP法进行软骨基质主要成分Ⅱ型胶原的免疫组化染色(抗体浓度1∶200)。采集图像后，在软骨区域随机选取3个视野，计算髁突软骨中Ⅱ型胶原阳性面积百分比。
1.7 TUNEL染色
石蜡切片常规脱蜡至水，按照TUNEL试剂盒说明书操作。蛋白K工作液室温下孵育20 min，PBS洗3次；TUNEL反应液37 ℃下避光孵育90 min，PBS洗3次；滴加含DAPI的抗荧光淬灭封片剂进行封片，4 ℃避光保存。聚焦激光扫描显微镜采集图像，在软骨区域随机选取3个视野，计算TUNEL阳性细胞率。
1.8 Western blotting检测
取右侧髁突软骨，冷冻捣碎后使用Tripure裂解液裂解并提取总蛋白，采用BCA法进行蛋白定量。聚丙烯酰胺凝胶电泳分离蛋白，半干电转仪转膜后封闭1 h，加入一抗(TNF-α抗体1∶500，cleaved-CASP3抗体1∶1000)，4 ℃孵育过夜。次日加入辣根过氧化物酶标记的羊抗小鼠IgG室温孵育1 h，加入化学发光液曝光显影。采集图像后采用Image Lab 5.2.1软件进行分析。
1.9 统计学处理
采用SPSS 21.0软件进行统计分析。计量资料均满足正态分布和方差齐性，以$\bar{x}±s$表示，多组间比较采用单因素方差分析，进一步两两比较采用Tukey检验。P<0.05为差异有统计学意义。
2 结果
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2.1 SHED治疗对MIA诱导所致TMJ髁突软骨退变的影响
2.1.1 HE染色结果
2、4周时，对照组TMJ髁突软骨各层(纤维层、增殖层、前肥大层、肥大层)排列规则，层次清晰，肥大层占软骨总厚度的50%左右。MIA组四层软骨严重不连续，层次紊乱，部分肥大层中断，软骨中出现大量无细胞区，基质网络紊乱，且髁突软骨纤维层较对照组和SHED组明显增厚，改良Mankin组织学评分明显高于对照组和SHED组，差异均有统计学意义(P<0.001)。SHED组髁突软骨尽管仍然存在软骨各层细胞界限不明及少量无细胞区，但形态基本恢复正常，其纤维层厚度、改良Mankin组织学评分与对照组比较差异无统计学意义(图1)。
2.1.2 番红O-固绿染色结果
TMJ髁突番红O-固绿染色显示，2、4周时，对照组番红O染色阳性的蛋白多糖均匀、规则地分布于软骨肥大层及前肥大层，少量分布于增殖层，且与固绿着色的纤维层、增殖层及软骨下骨分界清晰；MIA组髁突软骨中番红O着色、分布不均匀，不规则排列的软骨细胞染色严重缺失，周边染色增强，且番红O染色阳性面积百分比明显低于对照组(P<0.001)；SHED组番红O染色基本恢复正常，2周时番红O染色阳性面积百分比与对照组比较差异无统计学意义(P>0.05，图2)。
2.1.3 Ⅱ型胶原免疫组化染色结果
免疫组化染色显示，2、4周时，对照组Ⅱ型胶原阳性细胞染色均匀、规则，主要分布于肥大层及前肥大层；MIA组Ⅱ型胶原着色、分布不均匀，且Ⅱ型胶原阳性面积百分比明显低于对照组(P<0.01)；SHED组的Ⅱ型胶原着色基本恢复正常，Ⅱ型胶原阳性面积百分比明显高于MIA组(P<0.01)，而4周时与对照组比较差异无统计学意义(P>0.05，图3)。
2.2 SHED治疗对MIA诱导所致TMJ髁突软骨细胞凋亡的影响
TUNEL染色结果显示，2、4周时，对照组中几乎未见TUNEL阳性的死亡细胞；MIA组髁突软骨中TUNEL阳性细胞大量出现在增殖层和肥大层，其阳性细胞率明显高于对照组(P<0.001)；SHED组髁突软骨中TUNEL阳性细胞率与MIA组比较明显降低(P<0.001)，与对照组比较差异也有统计学意义(2周时P<0.01，4周时P<0.001，图4)。
2.3 SHED治疗对TMJOA软骨中cleaved-CASP3和TNF-α水平的影响
Western blotting检测结果显示，2、4周时，与对照组比较，MIA组TMJ髁突软骨中cleaved-CASP3和TNF-α蛋白表达量明显上调(P<0.001)；而经SHED治疗后，SHED组TMJ髁突软骨中的TNF-α和cleaved-CASP3蛋白表达量较MIA组明显下降(P<0.001)，而2周时TNF-α蛋白表达量与对照组比较差异无统计学意义(P>0.05，图5)。
3 讨论
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TMJOA是口腔领域第4大疾病TMD的重症表现形式，其致病机制截至目前尚不完全清楚，构建TMJOA动物模型是探索其机制的关键环节之一。当前诱导TMJOA的动物模型不少，主要分为化学药物诱导、机械应激刺激、外科手术诱导和心理应激诱导等4类[13]。TMJ关节腔内注射MIA诱导骨关节炎样病变已被广泛用于诱发TMJOA[14]。MIA主要通过抑制3-磷酸甘油醛脱氢酶的活性，使软骨细胞发生凋亡，进而诱导软骨退变。MIA可在TMJ中引起典型的OA样病变，软骨破坏呈时间依赖性，早期软骨细胞发生凋亡，后期为软骨基质紊乱，软骨下骨质侵蚀、纤维化，软骨下骨硬化，晚期则是骨赘形成[15]。MIA腔内注射可建立一种技术可靠、病变明显、操作简便的TMJOA大鼠模型，为TMJOA的相关研究提供参考。本研究MIA造模后TMJ髁突软骨层次紊乱，局部软骨基质丢失，并出现大量无细胞区，改良Mankin组织学评分也明显增高，提示TMJ上腔内注射MIA成功诱导了TMJOA样变，该模型有效、可靠。
近年来，干细胞疗法的开发和建立为有效治疗TMJOA提供了新的希望。SHED是一种具有高度增殖能力的克隆形成细胞群，具有明显的多向分化潜能[16]。与骨髓等其他来源的间充质干细胞相比，SHED具有与颅面组织相同的胚胎学来源和相似的基因表达模式，与颅面组织具有更高的亲和力。与牙髓间充质干细胞相比，SHED具有更高的增殖活性和成骨分化潜能[10]。2019年Luo等[17]采用SHED的外泌体(SHED-Exos)或SHED细胞上清(SHED-CM)预处理软骨细胞，然后用10 ng/ml白细胞介素(IL)-1β刺激，提取细胞裂解物后进行检测分析，结果发现SHED-Exos可通过miR-100-5p抑制哺乳动物雷帕霉素靶蛋白(mTOR)，进而抑制IL-6、IL-8、基质金属蛋白酶1(MMP-1)、MMP-3、MMP-9、MMP-13及血小板反应蛋白解整合素金属肽酶5(ADAMTS5)等促炎降解因子的表达。但该研究仅涉及体外实验，缺乏体内实验的验证。2020年Ogasawara等[18]经尾静脉注射SHED无血清条件培养液(SHED-CM)治疗强迫张口诱导的TMJOA模型小鼠，结果发现SHED-CM可通过下调IL-1β、诱导型一氧化氮合酶(iNOS)和MMP-13等促炎降解因子，特异性增加PCNA抗原阳性细胞数量，并改善TMJOA髁突软骨的完整性和表面光滑度，从而促进TMJ的再生修复。尾静脉注射一方面不易将发挥作用的起效细胞/因子精准靶向于目标位点，另一方面静脉注射外源性干细胞或相关物质对全身的影响未知，而局部注射较传统的全身静脉注射的药物剂量更低，引起的全身和局部不良反应更少，因此，关节腔内局部注射的针对性更强，不良反应更少。本研究发现，MIA造模后TMJ髁突软骨层次严重紊乱，部分肥大层中断，局部软骨基质(蛋白多糖、Ⅱ型胶原)大量丢失；SHED治疗后TMJ髁突软骨形态趋于正常，软骨基质丢失得到明显逆转。因此，SHED治疗可明显减少髁突软骨细胞外基质的降解，基本恢复软骨的形态。
软骨细胞是髁突软骨中唯一的细胞类型，软骨细胞凋亡是MIA所致OA早期软骨的重要病理表现[19-20]。本研究TUNEL染色显示，MIA组髁突软骨中TUNEL阳性细胞数目明显增多，细胞凋亡关键蛋白cleaved-CASP3的表达水平也明显升高，而SHED治疗后髁突软骨中TUNEL阳性细胞数目减少，cleaved-CASP3表达水平明显降低，提示SHED细胞悬液可有效抑制MIA所致的髁突软骨细胞凋亡。此外，TNF-α是髁突软骨退变过程中的关键炎性因子之一[21-22]，已被证实可抑制软骨细胞中蛋白多糖、连接蛋白和Ⅱ型胶原的合成[23]。本研究发现，MIA组髁突软骨中TNF-α蛋白表达水平明显升高，而在SHED治疗后即明显降低，提示SHED细胞悬液可明显抑制髁突退变软骨中TNF-α等炎性因子的表达。
综上，本研究发现，TMJ腔内局部注射SHED细胞悬液可减少软骨细胞外基质的降解、抑制软骨细胞早期凋亡、下调退变软骨中TNF-α等炎性因子的表达，最终有效逆转TMJOA进程中髁突软骨的退变，为临床治疗TMJOA提供了一种新策略。但本研究仍存在一定的局限性：TUNEL染色时未行大鼠软骨细胞标志物的共染色，未能进一步对比说明；只检测了髁突软骨中cleaved caspase 3的表达水平，而未检测总caspase 3的表达水平；未检测关节液中TNF-α的表达水平。因此，SHED细胞悬液治疗TMJOA的具体作用机制仍需进一步研究。
基金
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  • 国家自然科学基金(81970953)
  • 国家自然科学基金(82271000)
  • 陕西省自然科学基础研究计划重点项目(2022JZ-52)
文献
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参考文献 引证文献
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2023年第48卷第8期
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doi: 10.11855/j.issn.0577-7402.2338.2023.0601
  • 接收时间：2022-11-09
  • 首发时间：2025-11-25
  • 出版时间：2023-08-28
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  • 收稿日期：2022-11-09
  • 录用日期：2023-01-09
基金
National Natural Science Foundation of China(81970953)
国家自然科学基金(81970953)
National Natural Science Foundation of China(82271000)
国家自然科学基金(82271000)
Key Project of Shaanxi Provincial Natural Science Basic Research Program(2022JZ-52)
陕西省自然科学基础研究计划重点项目(2022JZ-52)
作者信息
    1安徽医科大学空军临床学院/安徽医科大学第五临床医学院/空军特色医学中心口腔科，北京 100142
    2空军军医大学口腔医院解剖生理学教研室/军事口腔医学国家重点实验室/口腔疾病国家临床医学研究中心/陕西省口腔疾病国际联合研究中心，陕西西安 710032

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2种不同金属材料的力学参数
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genus		 种数
Number of
species		 占总种数比例
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total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
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鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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