脂肪基质血管组分对兔跟腱损伤修复的研究

doi:10.3877/cma.j.issn.2095-1221.2021.04.006

目的

探讨肌腱内注射脂肪来源的基质血管组分（SVF）对兔跟腱损伤的修复作用。

方法

采用前列腺素E2诱导兔跟腱损伤模型，造模组注射前列腺素E2，空白对照组注射等剂量的生理盐水，超声检查、HE染色和Masson染色评估造模情况。损伤模型成模后，将造模组随机分为2组，SVF治疗组于跟腱局部注射兔子来源的脂肪SVF，模型对照组注射等剂量的生理盐水，分别于治疗后第3、8周，与空白对照组共同进行超声检查，并取跟腱组织标本行组织学检查及免疫组化测定Ⅰ型、Ⅲ型胶原的表达，多组间比较采用单因素方差分析，进一步通过LSD进行组间多重比较。

结果

前列腺素E2诱导后，超声结果显示肌腱局部肿胀，回声强度不均匀；HE染色及Masson染色结果可见明显的纤维断裂、血管增生、炎症细胞浸润和脂肪变性。与模型对照组相比，SVF组治疗后第3、8周低回声区域缩小，纤维结构改善，炎症细胞减少，Ⅰ型（第3周：0.2948±0.0126比0.3186±0.0108，第8周：0.3127±0.0157比0.3427±0.0138）、Ⅲ型胶原蛋白表达含量（第3周：0.2815±0.0143比0.3025±0.0141，第8周：0.2981±0.0153比0.3213±0.0104）均升高，差异有统计学意义（P均< 0.05）。

结论

SVF能减轻肌腱的炎症反应，改善胶原纤维结构和形态，上调Ⅰ型、Ⅲ型胶原蛋白表达水平，促进兔跟腱损伤的愈合。

关键词: 基质血管组分, 脂肪干细胞, 肌腱, 前列腺素E2, 跟腱损伤

Objective

To investigate the effects of intratendinous injection of adipose-derived stromal vascular fraction (SVF) on the repair of rabbit Achilles tendon injury.

Methods

Rabbit Achilles tendon injury model was induced by prostaglandin E2 (PGE2) . The injury model group received PGE2 injections, while the non-injury control group was injected with equal doses of saline. Ultrasonography, HE staining and Masson staining were used to evaluate the modeling. And the model group were randomly divided into two groups. The SVF treatment group was injected with rabbit-derived adipose SVF locally in the Achilles tendon, and the model control group was injected with equal doses of saline, respectively. Then, at 3 and 8 weeks after treatment, ultrasonography examination was performed. Achilles tendon tissue samples were obtained and examined histologically. Expression of collagen I and III was determined immunohistochemically. One-way ANOVA was used between multiple groups, with further multiple comparisons between groups performed by LSD.

Results

Ultrasound results revealed that tendon tissue had localized swelling and heterogeneous internal echoes after the induction of PGE2. The results of HE and Masson staining revealed visible fiber fracture, vascular proliferation, inflammatory cell infiltration, and fatty degeneration. Compared with model control group, the hypoechogenicity and inflammatory cells were reduced, the structure of fibers was improved and inflammatory cells were reduced in SVF group was at 3 and 8 weeks after treatment, and the expression of collagen I (week 3: 0.2948 ± 0.0126 vs 0.3186 ± 0.0108, week 8: 0.3127 ± 0.0157 vs 0.3427 ± 0.0138) , collagen III (week 3: 0.2815 ± 0.0143 vs 0.3025 ± 0.0141, week 8: 0.2981 ± 0.0153 vs 0.3213 ± 0.0104) was all increased. All differences were considered statistically significant (all P < 0.05) .

Conclusions

SVF can reduce the inflammatory reaction of tendon, improve the structure and morphology of collagen fiber, up-regulate the expression level of collagen I and III, and promote the healing of Achilles tendon injury in rabbits.

Key words: Stromal vascular fraction, Adipose stem cells, Tendon, Prostaglandin E2, Achilles tendon injury

图1 肌腱造模效果的常规超声图像对比

图3 正置光学显微镜观察两组造模情况（Masson染色，×100）。a图为空白对照组，b图为造模组；空白对照组纤维结构完好，排列整齐；造模组纤维结构紊乱，断裂明显，多处不规则卷曲

图4 二维超声观察治疗后各组跟腱组织

图5 彩色多普勒超声观察治疗后各组跟腱组织血流

图6 正置光学显微镜观察各组跟腱组织的治疗结果（HE染色，×100）

图7 正置光学显微镜观察各组跟腱组织的治疗结果（Masson染色，×100）

图8 正置光学显微镜观察各组跟腱组织的Ⅰ型胶原表达情况（免疫组化染色，×200）

图9 正置光学显微镜观察各组跟腱组织的Ⅲ型胶原表达情况（免疫组化染色，×200）

表1 治疗后各组跟腱组织的Ⅰ型、Ⅲ型胶原蛋白表达水平（ ± s）

分组	动物数	Ⅰ型胶原		Ⅲ型胶原
分组	6	3周	8周	3周	8周
空白对照组	6	0.3948±0.0112^b	0.3934±0.0107^b	0.3590±0.0123^b	0.3572±0.0115^b
模型对照组	6	0.2948±0.0126^a	0.3127±0.0157^a	0.2815±0.0143^a	0.2981±0.0153^a
SVF治疗组	6	0.3186±0.0108^ab	0.3427±0.0138^ab	0.3025±0.0141^ab	0.3213±0.0104^ab
F值		102.406	43.746	45.085	28.000
P值		< 0.001	< 0.001	< 0.001	< 0.001

表1 治疗后各组跟腱组织的Ⅰ型、Ⅲ型胶原蛋白表达水平（ ± s）

分组	动物数	Ⅰ型胶原		Ⅲ型胶原
分组	6	3周	8周	3周	8周
空白对照组	6	0.3948±0.0112^b	0.3934±0.0107^b	0.3590±0.0123^b	0.3572±0.0115^b
模型对照组	6	0.2948±0.0126^a	0.3127±0.0157^a	0.2815±0.0143^a	0.2981±0.0153^a
SVF治疗组	6	0.3186±0.0108^ab	0.3427±0.0138^ab	0.3025±0.0141^ab	0.3213±0.0104^ab
F值		102.406	43.746	45.085	28.000
P值		< 0.001	< 0.001	< 0.001	< 0.001

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Repairment of anchilles tendon injury by adipose derived stromal vascular fraction in rabbits

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Repairment of anchilles tendon injury by adipose derived stromal vascular fraction in rabbits