目的研究间充质干细胞（MSCs）对手术法诱导的大鼠膝骨关节炎（KOA）的治疗作用及其在裸大鼠和食蟹猴体内的组织分布及定植分化。方法 （1） Lewis大鼠随机分为假手术组、模型组、玻璃酸钠（每关节腔0.5 mg）组和MSCs低、中、高剂量（每关节腔3.0× 10⁵、1.0× 10⁶、3.0× 10⁶个细胞，其中高剂量为临床拟用剂量的等效剂量）组，除假手术组外，其余每组大鼠均右侧膝关节单侧手术法造模。使用X射线机检测大鼠膝关节病变；给药后记录大鼠痛阈变化；剖杀时记录关节软骨大体观察评分；试剂盒法检测血清中白细胞介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）、胶原蛋白Ⅱ型（Col Ⅱ）、Ⅱ型前胶原羧基端肽（PⅡCP）水平；取膝关节组织进行病理切片，苏木素-伊红（HE）染色观察组织损伤。（2）分别采用裸大鼠及食蟹猴进行MSCs在体内的组织分布及定植分化研究，实时荧光定量PCR（qRT-PCR）法检测供试品在裸大鼠、食蟹猴体内的组织分布，免疫组化法检测供试品在裸大鼠体内阳性分布的脏器组织。结果 （1）与假手术组相比，模型组X光评分、关节软骨大体评分、组织病理学评分、痛阈值显著升高，血清中IL-6、TNF-α含量明显升高，Col Ⅱ、PⅡCP含量明显降低（P＜0.01）；与模型组相比，MSCs可不同程度改善上述变化，其中高剂量组差异显著（P＜0.05、0.01）。（2）溶媒对照组裸大鼠及食蟹猴的全血及组织样品中均未检测到可定量的供试品细胞，裸大鼠末次给药后24 h、60 d、150 d仅在右侧膝关节组织中检测到可定量的供试品细胞，但不存在MSCs定植。食蟹猴末次给药后24 h在半月板中检测到可定量的供试品细胞，末次给药后24 h、3 d仅在膝关节腔冲洗液中检测到可定量的供试品细胞，且随观察期延长呈递减趋势。结论 MSCs每2周1次关节腔内注射对手术诱导的大鼠KOA有明显的治疗作用，其作用基于局部抗炎与软骨保护，且干细胞主要局限于给药关节组织内，不向全身其他组织分布或形成异位定植。

Objective To investigate the therapeutic effect of mesenchymal stem cell (MSCs) on surgically induced knee osteoarthritis in rats, as well as the tissue distribution and engraftment differentiation of stem cells in nude rats and cynomolgus macaques. Methods (1) Lewis rats were randomly divided into sham-operated group, model group, sodium hyaluronate group (0.5 mg per joint cavity), and low-, medium-, and high-dose MSCs groups (3.0×10⁵, 1.0×10⁶, and 3.0×10⁶ cells per joint cavity, respectively, with the high dose being an equivalent clinical dosage). Except for the sham group, all other groups underwent unilateral surgical induction of knee osteoarthritis in the right knee. Radiographic evaluation was performed to assess knee joint lesions; pain thresholds were recorded after treatment; gross scoring of articular cartilage was conducted at necropsy; serum levels of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), type II collagen (Col II), and type II procollagen carboxy-terminal peptide (PⅡCP) were measured using enzyme-linked immunosorbent assay (ELISA); knee joint tissues were subjected to histopathological examination via hematoxylin-eosin (HE) staining to evaluate tissue damage. (2) Tissue distribution and engraftment differentiation of MSCs in vivo were studied in nude rats and cynomolgus monkeys. Real-time quantitative PCR (qRT-PCR) was used to detect the distribution of test cells in various tissues of nude rats and cynomolgus monkeys; immunohistochemistry was employed to identify positive organ tissues with MSCs in nude rats. Results: (1) Compared with the sham group, the model group showed significantly higher X-ray scores, gross cartilage scores, histopathological scores, and pain thresholds, along with markedly elevated serum IL-6 and TNF-α levels and significantly reduced Col II and PⅡCP levels (P < 0.01). Compared with the model group, MSCs improved these parameters to varying degrees, with significant differences observed in the high-dose group (P < 0.05, 0.01). (2) No quantifiable test cells were detected in whole blood or tissues of solvent control groups in either nude rats or cynomolgus monkeys. In nude rats, quantifiable test cells were detected only in the right knee joint tissue 24 h, 60 d, and 150 d after the final administration, but no MSC engraftment occurred. In cynomolgus monkeys, quantifiable test cells were detected in the meniscus 24 h after the final dose, and only in the synovial lavage fluid 24 h and 3 d post-administration, showing a decreasing trend over time. Conclusion Intrajoint injections of MSCs every two weeks significantly ameliorate surgically induced knee osteoarthritis in rats, primarily through local anti-inflammatory effects and cartilage protection. The stem cells remain largely confined to the injected joint tissue without spreading to other systemic organs or forming ectopic engraftments.

R965

天津市科技计划项目（25YFKFYS00270，24ZYCGSY00640）；全国重点实验室重大专项（ S26ZS03094）