葛根代謝物及糖尿病對大鼠骨髓間葉幹細胞之影響-體外試驗

Ex vivo Effects of Puerariae radix Metabolites on Mesenchymal Stem Cells Derived from Rat Bone Marrow of Diabetes Mellitus Rats

胡文愷(Wen-Kai Hu)

指導教授 ： 李文婷

中原大學/工學院/生物醫學工程研究所/碩士(2010年)

https://doi.org/10.6840/CYCU.2010.00016

摘要

本研究目的在探討糖尿病及葛根血清代謝物 (SMP) 對大鼠骨髓間葉幹細胞 (rbMSCs) 之影響。首先利用四氧嘧啶破壞大鼠胰臟 β 細胞建立第一型糖尿病大鼠模式，經雙能 X 光吸收分析結果發現糖尿病大鼠之脛骨與股骨的骨質含量、面積以及密度均較正常鼠低。進一步探討糖尿病對 rbMSCs 之影響，結果發現血糖過高使得 rbMSCs 增生下降 5~30 %，抑制直徑大於 0.6 cm 細胞群落產生，並使得胞內 ATP 含量下降。乳酸脫氫酶 (LDH) 釋放分析，結果發現糖尿病不會破壞細胞膜完整性而導致 LDH 釋出量提高，顯示高血糖不會造成細胞壞死。接著探討其對細胞凋亡之影響，結果發現血糖過高會造成細胞核皺縮，DNA 片段化等細胞凋亡現象。進一步分析糖尿病對細胞內氧化壓力之影響，發現糖尿病 rbMSCs 的 ROS含量增加 38~40 %，且高血糖或高葡萄糖環境下會降低超氧化物歧化酶 (SOD) 及過氧化氫酶 (CAT) 活性；推測血糖過高造成 ROS 增加，進而導致細胞凋亡。在糖尿病對 rbMSCs 的脂肪與硬骨分化能力影響方面，結果發現高血糖或高葡萄糖環境會促進 rbMSCs 脂肪誘導分化時之 PPARγ 基因表現及三酸甘油脂累積，降低在硬骨誘導分化時之鹼性磷酸酶活性及 RUNX2 基因表現。接著探討 SMP 對於 rbMSCs 之影響，結果發現添加 SMP 可使正常鼠 rbMSCs 生長增加 11 %、糖尿病鼠rbMSCs生長增加 13 %。促進 SOD 及 CAT 活性，減少高血糖與高葡萄糖環境下之細胞凋亡以及胞內 ROS 含量，且抑制 rbMSCs 脂肪分化時之 PPARγ 基因表現，並提升其在硬骨分化時之 RUNX2 及 BMP-2 基因表現。綜上所言，糖尿病會抑制 rbMSCs 的生長及硬骨分化能力，促進幹細胞凋亡表現及脂肪分化能力。添加 SMP 能夠增加糖尿病 rbMSCs 的生長及硬骨分化能力，提升細胞內抗氧化酶活性，並降低 rbMSCs 培養高葡萄糖環境下的胞內 ROS 含量、細胞凋亡以及脂肪分化能力。

關鍵字

活性氧分子 ； 葛根血清代謝物 ； 硬骨生成 ； 四氧嘧啶 ； 脂肪生成 ； 骨髓間葉幹細胞 ； 糖尿病 ； 血糖過高

並列摘要

This study was undertaken to investigate the effects of serum metabolites of the Pueraria lobata (SMP) and diabetes on rat bone marrow-derived mesenchymal stem cells (rbMSCs). Type I diabetes rat model with pancreas β cell dysfunction was established by using alloxan. Bone mineral content, bone mineral volume, and bone mineral density of tibias and femurs in diabetic rats were decreased compared to those of normal rats as shown by Dual energy X-ray absorptiometry. The effect of diabetes on rbMSCs was explored and the results showed that hyperglycemia led to an inhibition on cell proliferation by 5~30 % and the number of colony forming-fibroblast with diameter greater than 0.6 cm. Intracellular ATP level of diabetic rbMSCs was lower than that of control group. Lactate dehydrogenase (LDH) release analysis showed that hyperglycemia did not seem to affect membrane integrity. No apparent LDH release was observed, which suggested that hyperglycemia did not cause necrosis. The effect of hyperglycemia on rbMSCs apoptosis was then investigated. The phenomena of apoptosis such as DNA fragmentation and chromatin condensation were found in cells under high glucose condition. Hyperglycemia was associated with 38~40 % increase in reactive oxygen species (ROS) level, and had significantly down-regulated the activities of superoxide dismutase (SOD) and catalase (CAT), which suggested that ROS might be responsible for apoptosis. The effects of hyperglycemia and high glucose (HG) treatment on both osteogenic and adipogenic differentiation potentials of MSCs were next detected. The results showed that hyperglycemia and HG treatment caused increased mRNA expression of PPARγ and triacylglycerol accumulation in rbMSCs during adipogenesis, and reduced expression of RUNX2 mRNA and alkaline phosphatase (ALP) activity during osteogenesis. The effects of SMP on diabetic rbMSCs were further investigated. Treatment of rbMSCs with SMP was associated with 11 % increase of control group and 13% increase of diabetes group in cell proliferation. SMP treatment increased the activities of the anti-oxidative enzymes, SOD and CAT, and decreased apoptosis as well as intracellular ROS level induced by hyperglycemia and HG treatment in rbMSCs. SMP treatment also decreased PPARγ mRNA expression during adipogenesis and increased RUNX2 and BMP-2 mRNA expression during osteogenesis in rbMSCs. In conclusion, diabetes rbMSCs exhibited the inhibitory effects on cell growth and osteogenic ability. The apoptosis and adipogenic capability of rbMSCs were increased by hyperglycemia. Furthermore, SMP treatment enhanced cell proliferation, antioxidant enzyme acitivities and osteogenic ability of diabetic rbMSCs. SMP treatment also decreased intracellular ROS level, apoptosis, and adipogenic capability of rbMSCs with HG treatment.

並列關鍵字

reactive oxygen species ； alloxan ； serum metabolites of the Pueraria lobata ； osteogenesis ； adipogenesis ； bone marrow mesenchymal stem cell ； hyperglycemia ； diabetes mellitus

參考文獻

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