摘要
目的：
恶性肿瘤已成为危害人类健康的一大疾病,化疗现已成为肿瘤临床综合治疗的重要手段之一。化疗虽然可以杀伤患者体内残余肿瘤细胞,提高疗效,改善预后,但其副作用(骨髓抑制)是造成患者不能按时足量完成化疗,或导致患者发生严重感染而死亡的重要原因。现代医学对骨髓抑制的预防和治疗方法仍有许多局限性,因此,探讨中医药疗法防治恶性肿瘤化疗后骨髓抑制有重要的临床意义。本课题以“肾藏精,精生髓化血”理论为依据,在前期研究基础上,选择巴戟天的成分巴戟天多糖、何首乌的成分二苯乙烯苷作为补肾法的代表,观察巴戟天多糖、二苯乙烯苷对骨髓抑制的作用,并从骨髓干细胞角度探讨其机理。为中西医结合治疗各种恶性肿瘤化疗后骨髓抑制的康复提供科学的实验依据。
方法：
1、应用《中华医典》、《古今图书集成医部全录》等检索工具,对“’肾藏精”理论进行系统梳理,寻求中医“肾藏精”的功能与现代医学骨髓干细胞功能的共通性,为中医药对恶性肿瘤化疗后骨髓抑制的康复作用提供科学的理论依据。
2、实验研究
(1)造模：根据Lena A Basile等方法采用环磷酰胺(250mg/kg)腹腔注射一次性造模成功。
(2)分组：将实验小鼠平均分为4组,每组12只,分别为预处理组、处理组、骨髓抑制模型组、空白对照组。
(3)给药方法：预处理组第1至7天用药,处理组第8天用药,巴戟天多糖或二苯乙烯苷每天一次,灌胃。预处理组、处理组与模型组第8天环磷酰胺250mg/kg腹腔注射。空白对照组第1至第14天,每天一次灌等量生理盐水。
(4)指标检测：
①血常规：预处理组分别在第1天、8天、12天、15天取血；其余各组分别在第8天、第12天、第15天取血。
②细胞计数：采取显微镜细胞观察方法进行骨髓有核细胞计数。
③流式细胞仪检测骨髓干细胞C-kit、CD105阳性细胞的表达,细胞周期。
④ELESA法检测小鼠粒细胞巨噬细胞集落刺激因子(GM-CSF)、小鼠干细胞因子／肥大细胞生长因子(SCF/MGF)。
结果：
1、巴戟天多糖：
(1)对骨髓抑制模型血成分的调控影响：红细胞到化疗后第7天模型组显著低于空白组,说明此时红细胞到达低点,但此时处理组与空白组比较无显著性差异,说明化疗后使用巴戟天多糖可以减缓骨髓抑制模型RBC的下降；HGB第12天模型组与空白组比较差异显著,但预处理组与空白组比较无显著性差异。
(2)对骨髓抑制模型骨髓干细胞增殖的影响：化疗后4天,预处理组与处理组CD117阳性细胞值显著高于空白组(P<0.05); CD105-CD117Double预处理组明显高于模型组(P<0.05)。化疗后4天取材,预处理组、处理组与模型组小鼠GO/Gl期骨髓细胞比例较空白组均显著下降。S期和G2/M期骨髓细胞比例较模型组均显著上升。而与处理组比较,预处理组小鼠G0/G1期骨髓细胞比例显著下降,S期骨髓细胞比例显著上升。
(3)对干细胞生长因子分泌的影响：预处理组血中的GM-CSF浓度显著高于模型组,差异有显著性(P<0.05)。
2、二苯乙烯苷：
(1)对骨髓抑制模型血成分的调控影响：化疗后第4天,除空白组外各组都显著降低,但预处理组的白细胞值显著高于处理组与模型组(P<0.05)；模型组RBC、HGB值较空白组显著下降(P<0.01),但处理组与空白组比较无显著性差异。
(2)对骨髓抑制模型骨髓干细胞增殖的影响：化疗后4天,预处理组与处理组CD117阳性细胞值显著高于空白组(P<0.05)；CD105阳性细胞值,与空白组相比,各组均显著增高(P<0.01),同时处理组显著高于预处理组(P<0.05)；化疗后4天取材,预处理组、处理组与模型组小鼠G0/G1期骨髓细胞比例较空白组均显著下降。S期和G2/M期骨髓细胞比例较模型组均显著上升,7天后回复至正常水平。
(3)对于细胞生长因子分泌的影响：预处理组血清中的SCF/MGF浓度与空白组、模型组、处理组比较,有显著性差异(P<0.05)。
结论：
1、巴戟天多糖处理对小鼠化疗后骨髓抑制有促进作用,可增加骨髓干细胞S期的比例,减少GOG1期比例；巴戟天多糖预处理可增加G-CSF的分泌：巴戟天多糖可增加骨髓干细胞CD105、CD117阳性细胞表达,促进骨髓干细胞增值,使外周血象的红细胞、血红蛋白数值增加,进而对化疗后骨髓抑制模型有积极的调控作用。
2、二苯乙烯苷预处理对小鼠化疗后骨髓抑制有促进作用,可增加骨髓干细胞CD117阳性细胞表达,增加SCF的分泌,使外周血象的白细胞数值增加,进而改善化疗的骨髓抑制的毒副作用。化疗后应用二苯乙烯苷处理可增加骨髓干细胞CD117、CD105阳性细胞表达,促进骨髓干细胞增值,从而增加外周血象的红细胞、血红蛋白数值,对化疗后骨髓抑制模型有积极的调控作用。
Objective
Malignant tumors have become a major kind of diseases which hazard to human health, chemotherapy has now become an important means of the comprehensive treatment in clinical oncology. Chemotherapy can kill and wound residual tumor cells in vivo, enhance the curative effect and improve the prognosis. But the side effect of chemotherapy (bone marrow suppression) is the main cause which makes patients unable to sufficiently complete chemotherapy on schedule, or makes patients die for serious infection. To bone marrow suppression, at present, western medicine hasn't effective prevention and treatment ways, for this reason, it has important clinical significance to probe into traditional chinese medicine treatment which can prevent bone marrow suppression after malignant tumor chemotherapy. This topic is based on the theory of "the kidney stores essence, and the essence can grow into marrow, then the marrow produces blood".According to the previous studies, Morinda officinalis How polysaccharide and stilbene glucoside were selected as the representative of tonify the Kidney, they were respectively from the Morinda officinalis How and Polygonum multiforum Thunb.Then the effect which these two ingredients act on bone marrow suppression was observed, then the mechanism of the effect was studied from the perspective of marrow stem cells. It provides science experimental evidence for using combination traditional Chinese and western medicine to treat the bone marrow suppression after chemotherapy in various malignant tumor
Methods
1、《Chinese Medical Classics》、《The Integration Of Ancient And Modern Medical Books All The Record》 etc search tools was applicated to systematically analyze the theory of "the kidney stores essence", for seeking the commonality between the function of "the kidney stores essence" in Traditional Chinese Medicine theory and the function of bone marrow stem cell in western medicine, also for providing science theoretical evidence for the bone marrow suppression recovery of using traditional Chinese medicine in malignant tumor after chemotherapy.
2、Experiment study
(1) Animal Model Making:According to the Lena A Basile method etc,the cyclophosphamide (250mg/kg) was used for intraperitoneal injection and then the model was successfully made at one-time.
(2) Grouping:Experimental mice were divided into four groups, they were pretreatment group、treatment group、bone marrow suppression model group、 blank control group, each group containing12.
(3) dosing:Preconditioning group were gavaged during the lst-7th day, treatment group were gavaged at the8th day, both team were gavaged once a day with the aqueous solution of Morinda officinalis How polysaccharide or THSG. The cyclophosphamide (250mg/kg) was used for intraperitoneal injection in preconditioning group、treatment group、bone marrow suppression model group. Blank control group were gavaged once a day with the same amount of saline.
(4) indicator testing:
①blood routine:The blood were took from preconditioning group when the1st、8th、12th、15th day and other group when the8th、12th、15th day.
②cell count:Using the microscope observed nucleated bone marrow cells,then counted.
③The expression of positive C-kit and CD105of bone marrow stem cells were tested by flow cytometry
④GM-CSF、SCF/MGF were tested by ELESA method.
Results
1、Morinda officinalis How polysaccharide
(1)The effect on regulation of blood composition of bone marrow suppression model group:The content of red blood cell in bone marrow suppression model group was significantly lower than it in blank control group after chemotherapy at the7th day, it means that red blood cell had been reached low point.But it had no significantly difference between treatment group and blank control group, it showed that Morinda officinalis How polysaccharide could slow down the decline of the RBC in bone marrow suppression model group after chemotherapy. It also had been found that the HGB in bone marrow suppression model group was significantly different from blank control group at the12th day, but it had no significantly difference between pretreatment group and blank control group.
(2) The effect on proliferation of bone marrow stem cell in the bone marrow suppression model:After chemotherapy4days, the value of positive CD117in pretreatment group was significantly higher than it in treatment group(P<0.05), while CD105-CD117Double pretreatment group was higher than bone marrow suppression model group (P<0.05). Getting specimen after chemotherapy4days, the bone marrow cells proportion of G0/G1decreased significantly in pretreatment group, treatment group and bone marrow suppression model group compared with blank control group. The proportion of bone marrow cells in phase S and G2/M increased significantly than bone marrow suppression model group, while the proportion of G0/G1in pretreatment group decreased significantly and increased significantly in phase S compared with treatment group.
(3) The effect on secretion of stem cell growth factor:The concentration of GM-CSF in pretreatment group was significantly higher than bone marrow suppression model group, the difference was significant (P<0.05)
2、Stilbene Glucoside
(1) The effect on regulation of blood composition of bone marrow suppression model group:All groups descended significantly except blank control group, at the4th day after chemotherapy, but the value of white blood cells of pretreatment group were significantly higher than it in treatment group and marrow suppression model group (P<0.05). The value of RBC and HGB significantly descended than blank control group (P<0.01), but it had no significant difference between pretreatment group and blank control group.
(2) The effect on proliferation of bone marrow stem cell in the bone marrow suppression model:the value of positive CD117in pretreatment group and treatment group was significant higher than it in blank control group (P<0.05), at the4th day after chemotherapy;The value of positive CD105increased in all groups compared with blank control group, while the value of treatment group was significant higher than pretreatment group's (P<0.05); Getting specimen after chemotherapy4days, the bone marrow cells proportion of G0/G1descended significantly in pretreatment group, treatment group and bone marrow suppression model group compared with blank control group.The proportion of bone marrow cells in phase S and G2/M significantly increased than bone marrow suppression model group, while the proportion of G0/G1in pretreatment group decreased significantly and increased significantly in phase S compared with treatment group, and then returned to normal level after7days.
(3) The effect on secretion of stem cell growth factor:The concentration of SCF/MGF in pretreatment group was significantly different from blank control group, bone marrow suppression model group and treatment group (P<0.05)
Conclusion:
1.Morinda officinalis how polysaccharide has the promoting effect on mice which had been in bone marrow suppression after chemotherapy. It increases the proportion of S and the G2/M phase bone marrow stem cells, enhances the number of stem cells in bone marrow, promotes the secretion of G-CSF, and increases red blood cells and hemoglobin of the peripheral blood cells, and then improves the toxic and side effects of bone marrow suppression after chemotherapy.
2. Stilbene Glucoside has the promoting effect on mice which has been in bone marrow suppression after chemotherapy, it also increases the expression of positive CD117of bone marrow stem cells, the secretion of G-CSF, and the white blood cells of the peripheral blood cells, and then improves the toxic and side effects of bone marrow suppression after chemotherapy. Using Stilbene Glucoside after chemotherapy can increase the expression of positive CD117and CD105of bone marrow stem cells, promote the proliferation of bone marrow stem cells, and then increase the red blood cells、hemoglobin. It has positive regulation function to bone marrow suppression after chemotherapy after chemotherapy.
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