Treating deep vein thrombosis with human umbilical cord mesenchymal stem cell transplantation

doi:10.3969/j.issn.2095-4344.1745

Abstract

Abstract:

BACKGROUND: Human umbilical cord mesenchymal stem cells can home to ischemic sites and form functional blood vessels locally. Neovascularization is critical for the organization and recanalization of deep vein thrombosis. However, transplantation of human umbilical cord mesenchymal stem cells for the treatment of thrombosis has not been reported.
OBJECTIVE: To investigate the dissolution, organization and recanalization of deep vein thrombosis after the transplantation of human umbilical cord mesenchymal stem cells.
METHODS: Deep vein thrombosis model was established by “narrow method” in rats. Suspension of the third generation human umbilical cord mesenchymal stem cells at a density of 2×10⁹ cells/L was prepared. The model rats were randomly divided into four groups. In control group, 1 mL of PBS was injected into the tail vein on days 1, 2, and 3 after modeling. In experimental group 1, 1 mL of cell suspension was injected into the tail vein on day 1 after modeling, and 1 mL of PBS was injected on days 2 and 3. In experimental group 2, 1 mL of cell suspension was injected on days 1 and 2 after modeling, and 1 mL of PBS was injected on day 3. In experimental group 3, 1 mL of cell suspension was injected on days 1, 2, and 3 after modeling. The experimental protocol was approved by the Animal Experimental Ethics Committee of Nantong University with the approval No. 20180315-001 on April 1, 2018. Samples from deep vein thrombosis rats were taken on day 7 after modeling. Number of new blood vessels and dissolution rate in each group were detected by hematoxylin-eosin staining and immunohistochemical staining.
RESULTS AND CONCLUSION: Compared with the control group, the capillary density of the experimental groups 2 and 3 was significantly increased (P < 0.01). The dissolution rate of all the experimental groups was higher than that of the control group, but the difference was not statistically significant (P > 0.05). All these results reveal that a certain amount of human umbilical cord mesenchymal stem cells transplanted can increase the number of microvessels in the thrombosis, and accelerate organization and recanalization of the thrombosis.

Key words: deep vein thrombosis, organization, recanalization, human umbilical cord mesenchymal stem cells, angiogenesis, vascular density, dissolution rate, inferior vena cava thrombosis model, rat, stem cells

CLC Number:

R453
R394.2

Wang Yan, Shi Qin, Zhang Yuquan. Treating deep vein thrombosis with human umbilical cord mesenchymal stem cell transplantation[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(21): 3392-3397.

Figures/Tables 2

2.1 实验动物数量分析对照组在术后第2天死亡1只，实验组1其中1只在注射干细胞时，出现注射完毕即反应差、随后呼吸心跳骤停的情况，考虑系该次注射前未混匀细胞悬液，细胞团造成大鼠肺栓塞，后每次注射细胞前均使用口径较大的吸管轻柔吹打、混匀，未再次出现此类情况。最终18只大鼠进入结果分析。 2.2 人脐带间充质干细胞培养结果及其形态学特点培养三四天可见少量细胞从组织边缘爬出，多呈圆点状，见图2A；培养6 d进一步增多；培养8 d后见爬出明显增多，细胞由原来的圆状变成短棒状、有突起的长梭状，见图2B；培养13 d后细胞已有部分成片聚集，细胞多呈长梭状，偶扁平形的成纤维样细胞状，此时去掉组织块；培养16 d细胞浓度融合达80%-90%，低倍镜下见呈旋涡生长，见图2C。传代后细胞增长明显加快，P1代需每隔三四天换液1次，8 d后需1∶3传代。P2代细胞培养两三天即需换液，到第6天后需1∶4传代。若细胞浓度较低时常呈扁平样、多角样；若细胞浓度较高，趋于融合，常在低倍镜下呈旋涡状生长，高倍镜下可见细胞呈长梭形的成纤维细胞样。 2.3 苏木精-伊红染色结果及各组血栓溶解率的比较苏木精-伊红染色切片40倍镜下可见，有核炎性细胞由周边向内部浸润，机化由血栓周边开始。对照组：偶在血栓与血管壁之间见少量溶解裂隙，血栓内基本无溶解，血栓基本充满管腔；实验组1：血栓与血管壁之间裂隙较对照组稍有增加，血栓内溶解仍有限；实验组2：血栓与血管壁之间以及血栓内溶解显著增加，较对照组及实验组1明显；实验组3：血栓周围及内部的溶解进一步增加，见图3。各组血栓溶解率：细胞移植组较对照组增加，实验组3大于实验组2大于实验组1，但差异无显著性意义(P=0.077)，见表1。 2.4 各组血栓内新生血管数的比较 CD34免疫组织化学染色显示，低倍镜下小血管多位于血栓周边，细胞移植组的血栓内新生血管数量明显多于对照组，见图4。高倍镜下可见被染成棕褐色的管腔样结构的纵切面及横切面，且部分管腔内可见红细胞，见图5。各组平均血管密度：不同处理组间血栓的血管密度差异有显著性意义(P < 0.01)；组间两两比较示实验组1的平均血管密度比对照组高1.00(个/ 400倍视野)(95%CI：-2.23-4.23)，差异无显著性意义(P= 0.810)；实验组2的平均血管密度比对照组高3.65(个/ 400倍视野)(95%CI：0.59-6.71)，差异有显著性意义(P=0.018)；实验组3的平均血管密度比对照组高6.35(个/400倍视野)(95%CI：3.29-9.41)，差异有显著性意义(P < 0.01)；实验组3的平均血管密度比实验组2高2.70(个/400倍视野)(95%CI：-0.19-5.59)，差异无显著性意义(P= 0.07)，见表1。"

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