人類臍帶間質幹細胞對葡聚糖硫酸鈉誘發之小鼠急性及慢性結腸炎腸道內及腸道外表現的療效

Therapeutic effects of human umbilical cord mesenchymal stem cells on intra-intestinal and extra-intestinal manifestations on dextran sulfate sodium induced acute and chronic colitis in mice

張裕隆(Yu-Lung Chang)

指導教授 ： 謝明發

中原大學/工學院/生物醫學工程研究所/博士(2019年)

https://doi.org/10.6840/cycu201900514

摘要

目的: 文獻報導多次注射骨髓間質幹細胞用於治療小鼠的慢性結腸炎，特別是腹膜內(IP)注射間質幹細胞可以治療小鼠結腸炎，但是腹部器官損傷和感染等併發症是一個有待解決的問題。潰瘍性結腸炎與多種腸道外表現有關，如關節、皮膚、眼部和口腔表現、骨質疏鬆症、肝膽疾病、澱粉樣變性和睾丸損傷。本研究評估單次注射人類臍帶間質幹細胞（hUCMSCs）對葡聚糖硫酸鈉（DSS）誘導的急性和慢性結腸炎腸道內和腸道外(例如:睾丸損傷)表現的治療效果。
方法: 在單次注射hUCMSCs對小鼠急性和慢性結腸炎的治療試驗中，將雄性C57BL/6JNarl小鼠分成對照組、磷酸鹽緩衝鹽水組（PBS）和hUCMSCs注射組。在經由皮下（SC）或IP途徑單次注射hUCMSCs對小鼠結腸炎的治療試驗中，將雄性C57BL/6Ncr1小鼠分成對照組、PBS、hUCMSCs SC組及hUCMSCs IP組。在評估注射hUCMSCs對結腸炎小鼠睾丸損傷的治療試驗中，根據對照組老鼠的最低副睾精子數，將PBS和hUCMSCs組再細分為第一磷酸鹽緩衝鹽水組（PBS1）(睾丸功能障礙)、第二磷酸鹽緩衝鹽水組（PBS2）(正常睾丸功能)、第一hUCMSCs組（SC1）(睾丸功能障礙)和第二hUCMSCs組(SC2)(正常睾丸功能)。使用3％ DSS在小鼠（對照組除外）中誘導急性和慢性結腸炎。實驗數據包含疾病活動指數、結腸長度、結腸組織學、結腸炎症評分、幹細胞於小鼠體內各器官的分布、血液內細胞因子濃度、結腸組織中細胞因子和趨化因子的免疫組織化學染色、許多器官中hUCMSCs的免疫組織化學染色、睾丸組織學、睾丸重量、副睾精子數、Johnsen評分、血液睾固酮水平和睾丸組織中3β-HSD的免疫組織化學染色皆被檢測及紀錄。
結果：在急性和慢性結腸炎的治療試驗中，hUCMSCs組小鼠的疾病活動指數顯著高於對照組，且均低於PBS組。在第8天和第25天，hUCMSCs組的結腸長度明顯更長，結腸炎症評分顯著低於PBS組，這代表單次注射幹細胞有治療效果。在第8天和第25天，hUCMSCs組小鼠血清中的細胞因子和趨化因子(IL17A、Gro-α、MIP-1α、MIP-2和eotaxin)都比PBS組的顯著降低。
在急性結腸炎的晚期(第9-10天)，SC組小鼠的疾病活動指數顯著低於IP組。與對照組相比，PBS組小鼠的結腸長度明顯縮短，結腸炎症評分顯著增高，這代表注射PBS對急性和慢性結腸炎沒有治療效果。對照組和SC或IP組之間的結腸長度和炎症評分沒有顯著差異。在第8天和第25天，SC組的IL17A和Gro-α表現均低於IP組。經由SC注射的hUCMSCs在皮下組織中可累積至第25天。
在結腸炎小鼠睾丸損傷的治療試驗中，幹細胞組的精子生成損傷比例在第8天和第25天低於PBS組（P <0.05）。在第8天和第25天，PBS2組的總體睾丸功能損傷分別比PBS1組低（P = 0.031, 0.031）。在第8天和第25天，SC2組的總體睾丸功能損傷分別比SC1組低（P = 0.031, 0.031）。在第8天和第25天，PBS1組的結腸炎分別比PBS2組嚴重（P = 0.016, 0.016）。在第8天和第25天，SC1組的結腸炎分別比SC2組嚴重（P = 0.016, 0.016）。
結論：單次注射hUCMSCs改善了DSS誘導的急性結腸炎，並減少了急性結腸炎進展為慢性結腸炎。通過SC或IP注射的hUCMSC改善了DSS誘導的急性結腸炎並減少了慢性結腸炎的進展。hUCMSCs通過SC注射引起的抗發炎作用優於IP注射。在人體臨床實務中，SC注射hUCMSCs比IP注射相對方便和安全。幹細胞可以降低睪丸損傷。急性腸炎臨床症狀嚴重的小鼠睪丸損傷較嚴重。雖經幹細胞治療後，臨床嚴重組小鼠睪丸損傷程度仍比輕症組小鼠明顯。

關鍵字

人類臍帶間質幹細胞 ； 結腸炎 ； 睪丸損傷

並列摘要

Purpose: Multiple injections of bone marrow mesenchymal stem cells (BMMSCs) have been used for treatment of chronic colitis in mice. Intraperitoneal (IP) injection of mesenchymal stem cells (MSCs) has been reported to treat colitis in mice. Complications such as abdominal organ injury and infection might be significant concerns. The literatures revealed that ulcerative colitis is associated with a variety of extra-intestinal manifestations such as joint, skin, ocular and oral manifestations, osteoporosis, hepatobiliary disease, amyloidosis and testicular injury. In this study we would like to investigate the therapeutic efficacy of a single injection of human umbilical cord mesenchymal stem cells (hUCMSCs) on intra-intestinal and extra-intestinal (eg. testicular injury) manifestations on dextran sulfate sodium (DSS) induced acute and chronic colitis.
Methods: For evaluation of therapeutic effects of a single injection of hUCMSCs on acute and chronic colitis in mice. Male C57BL/6JNarl mice were divided into control, phosphate-buffered saline (PBS), and hUCMSCs treated groups, respectively. For evaluation of therapeutic effects of a single injection of hUCMSCs via subcutaneous (SC) or IP route in colitis mice, male C57BL/6Ncrl mice were divided into control, PBS, hUCMSCs SC, and hUCMSCs IP groups. For evaluation of therapeutic effects of a single dose of human umbilical mesenchymal stem cells on testicular injury of mice with colitis, the PBS and hUCMSCs groups were divided to the phosphate-buffered saline 1 (PBS1)(testicular dysfunction), phosphate-buffered saline 2 (PBS2) (normal testicular function), hUCMSCs 1 (SC1) (testicular dysfunction) and hUCMSCs 2 (SC2) (normal testicular function) groups according to the lowest epididymal sperm count of control group. Acute and chronic colitis were induced in the mice (except control group) using 3% DSS. The mice in the hUCMSCs group underwent a single injection of hUCMSCs. The disease activity index (DAI), colon length, colon histology, colon inflammation score, distribution of stem cells in various organs of mice, and blood cytokine levels, immunohistochemistry stain of cytokine, chemokine in the colon tissue, immunohistochemistry stain of hUCMSCs in many organs, testis histology, testis weight, epididymal sperm count, Johnsen score, blood testosterone level and immunohistochemistry stain for the detection of 3β-HSD in the testis tissue were recorded.
Results: The DAI of mice was significantly higher in the hUCMSCs group than in the control group and lower than in the PBS group on all days. The colon length was significantly longer and the colon inflammation score was significantly lower in the hUCMSCs group than in the PBS group on the 8th and 25th days. This means that single injection of stem cells has a therapeutic effect. The serum cytokine and chemokines (IL17A, Gro-α, MIP-1α, MIP-2, and eotaxin) of mice were significantly lower in the hUCMSCs group than those in the PBS group on the 8th and 25th days.
The DAI of mice in the SC group was significantly lower than that in the IP group during late acute colitis (the 9th and 10th days). Compared with the control group, the colon length of mice was significantly shorter, and the colon inflammation score of mice was significantly higher in the PBS group. This means that the injection of PBS has no therapeutic effect on acute and chronic colitis. There was no significant difference in the colon length and inflammation score between the control group and the SC or IP group. The expressions of IL17A and Gro-α decreased in the SC group compared with those in the IP group on the 8th and 25th days. hUCMSCs via SC injection accumulate in the subcutaneous tissue to the 25th day.
The ratio of spermatogenesis impairment in the stem cell groups was lower than that in the PBS groups on the 8th and 25th days (P<0.05). The overall testicular function impairment in the PBS2 group was lower than that in the PBS1 group on the 8th and 25th days, respectively (P=0.031, 0.031). The overall testicular function impairment in the SC2 group was lower than that in the SC1 group on the 8th and 25th days, respectively (P=0.031, 0.031). The severity of colitis in the PBS1 group was more severe than that in the PBS2 group on the 8th and 25th days, respectively (P=0.016, 0.016). The severity of colitis in the SC1 group was more severe than that in the SC2 group on the 8th and 25th days, respectively (P=0.016, 0.016).
Conclusions: Single-injection hUCMSCs improved DSS–induced acute colitis and decreased progression of acute colitis to chronic colitis. hUCMSCs via SC or IP injection improved DSS-induced acute colitis and decreased the progression to chronic colitis. The effect of anti-inflammation exerted by hUCMSCs via SC injection is better than that of IP injection. In clinical practice in human, SC injection of hUCMSCs is relatively convenient and safe than IP injection. Stem cells can reduce testicular damage. Mice with severe clinical symptoms on acute colitis have more severe testicular damage. Although after treatment with stem cells, the degree of testicular damage in the clinically severe group of mice was still significantly higher than that in the mild group.

並列關鍵字

human umbilical cord mesenchymal stem cells ； colitis ； testicular injury

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