人參皂苷Rg1透過脾臟髓外造血改善環磷酰胺所誘導的骨髓毒性

Ginsenoside Rg1 improves bone marrow hematopoietic activity in cyclophosphamide-induced myelosuppression mice via extramedullary hematopoiesis of the spleen

陳飛鵬(Fei-Peng Chen)

指導教授 ： 張格東

國立屏東科技大學/農學院/生物科技研究所/碩士(2015年)

https://doi.org/10.6346/NPUST.2015.00239

摘要

環磷醯胺（cyclophosphamide）是用於治療癌症和免疫疾病常用的化學藥物。使用環磷醯胺會產生骨髓的細胞毒性，並導致患者的骨髓抑制 (myelosuppression) 和髓外造血（extramedullary hematopoiesis）。髓外造血是特定病理狀態下的代償作用，主因是骨髓微環境變成不適合幹細胞和前驅細胞生長與分化。髓外造血最常發生在脾臟和肝臟，並偶爾會出現在淋巴結與其它組織部位。Xu等人的研究，發現使用人參皂苷可以改善環磷醯胺所誘導之骨髓抑制小鼠的造血活性，我們認為骨髓造血活性的恢復亦會改善脾臟的髓外造血現象。因此本研究主旨在探討環磷醯胺誘導的骨髓抑制小鼠是否可以透過人參皂苷Rg1的治療改善其髓外造血的現象。我們參考臨床醫學的用藥劑量，透過腹腔注射給予小鼠環磷醯胺（50 mg /kg，i.p. 4天）誘導骨髓抑制，之後連續7天再給予人參皂苷Rg1（15mg/kg）。完成投藥後隨即犧牲小鼠，進行骨髓和脾臟細胞數的計
數、脾臟組織學的觀察，並利用流式細胞儀分析骨髓、脾臟與周邊血的造血幹細胞 (Lin-、c-Kit+、Sca-1+，LSK) 與前驅細胞c-Kit+ 細胞的增生活性和細胞凋亡。我們的研究顯示，在環磷醯胺加Rg1治療後之小鼠脾臟重量減輕，脾臟的LSK 與c-Kit+ 細胞數量亦減少，且減少之細胞非細胞凋亡所造成，然而脾臟的巨核細胞比例沒有下降，顯示人參皂苷Rg1的治療可以減緩髓外造血現象，但無法完全消彌髓外造血。再者，雖然脾臟的LSK 與c-Kit+ 細胞數量減少，但脾臟的c-Kit+ 細胞增生活性卻明顯上升；有趣的是，骨髓的LSK細胞數量明顯增加，但c-Kit+ 細胞的增生活性卻不變，且周邊血的c-Kit+ 細胞數量亦增加。當環磷醯胺與Rg1作用於脾臟切除之小鼠後，其骨髓和周邊血的LSK 與c-Kit+ 細胞都不會增加。此結果顯示，Rg1治療環磷醯胺所誘導之骨髓抑制小鼠，不僅能減緩小鼠脾臟髓外造血，亦可能透過改變脾臟微環境，促成c-Kit+細胞在脾臟增生與歸返，最終導致骨髓造血活性的提升。

關鍵字

人參皂苷Rg1 ； 環磷醯胺 ； 骨髓毒性 ； c-Kit+細胞, ； 脾臟切除

並列摘要

Cyclophosphamide is a common chemical medicine for the treatment of cancer and immune diseases. The usage of cyclophosphamide causes bone marrow cytotoxicity resulting in bone marrow suppression in the patients and extramedullary hematopoiesis. Extramedullary hematopoiesis is the compensation happened underneath some pathological conditions, mainly due to bone marrow failure for growth and differentiation of stem and/or precursor cells. Extramedullary hematopoiesis occurs most often in the spleen and liver, occasionally appearing in the lymph nodes and other tissues. Xu et al found that treatment of ginsenoside Rg1 can improve hematopoietic stem and progenitor cells in cyclophosphamide-induced myelosuppressive mice. We therefore suspect that the recovery of bone marrow hematopoietic activity accompanies a relief of extramedullary hematopoiesis in the spleen. We followed clinical doses indication and administered cyclophosphamide (50 mg/kg) to mice for 4 days by intraperitoneal injection. Then we injected mice with ginsenosides Rg1 (15mg / kg) intraperitoneally for another 7 days. After that, mice were sacrificed and total bone marrow and splenocytes were analyzed by histology and flow cytometry. Our results showed that the weight of spleen in cyclophosphamide-induced mice reduced after Rg1 treatment. The number of hematopoietic stem and progenitor cells (Lin-, c-Kit +, Sca-1 + ; LSK) in the spleen were also decreased but not as a result of apoptosis. However, the proportion of splenic megakaryocytes was unchanged. These data manifested that treatment of ginsenosides Rg1 in cyclophosphamide-induced mice was able to alleviate extramedullary hematopoiesis. Meantime, the proliferative activity of c-Kit + stem and progenitor cells was also significantly increased in the spleen, but not in bone marrow. We also found an increased population of c-Kit + cells in the peripheral blood. Interestingly, when we treated Rg1 in cyclophosphamide-induced mice without the spleen, the effect of Rg1 on bone marrow was suppressed. The results demonstrated that extramedullary hematopoiesis in the spleen induced by cyclophosphamide confers the functionality of Rg1.
Our findings also illustrate that treatment of Rg1 in cyclophosphamide-induced mice very likely changes the microenvironment of the spleen. Hence, Rg1 upregulates the proliferation and homing activity of c-Kit + stem and progenitor cells in the spleen that eventually leads to bone marrow hematopoietic improvement.

並列關鍵字

ginsenoside Rg1 ； myelosuppression ； cyclophosphamide ； c-Kit+ cell ； splenectomized

參考文獻

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