【作者】 高珊；

【导师】 付蓉； 邵宗鸿；

【作者基本信息】 天津医科大学 ， 内科学， 2010， 硕士

【摘要】 目的：体外培养和纯化多发性骨髓瘤(multiple myeloma, MM)患者骨髓来源成骨细胞,并鉴定其生物学性状,比较其与正常成人骨髓来源成骨细胞的增殖分化和成骨潜能。比较治疗MM的新型药物——蛋白酶体抑制剂(硼替佐米)及MM患者血清对成骨细胞成骨能力的影响,初步探讨硼替佐米对MM成骨细胞的作用及机制。方法：研究对象为MM患者15例,正常对照10名。采用骨髓培养法,体外培养和纯化MM患者成骨细胞,传代后观察细胞形态,测定其生长曲线、倍增时间,进行碱性磷酸酶染色(ALP)、Von Kossa钙结节染色、Ⅰ型胶原抗体免疫组化染色等鉴定其生物学性状,并比较其与正常成人骨髓来源成骨细胞的成骨潜能。在骨髓瘤患者成骨细胞组及正常组中分别加入硼替佐米及骨髓瘤患者血清,对比加入调控因子前后细胞生物学特性的变化。使用流式细胞术(FCM)检测骨髓瘤成骨细胞细胞膜抗体(CD34、CD138、CD45)的表达,采用酶联免疫吸附测定(ELISA)法测定血清中白细胞介素-7(IL-7)的水平,并采用逆转录聚合酶链反应(RT-PCR)方法检测各组培养细胞中骨形成蛋白2(BMP2)表达。结果：1.采用骨髓培养法,条件培养基中加入地塞米松、β-甘油磷酸钠和维生素C等条件因子,在MM患者骨髓中,可培养并纯化出成骨细胞。细胞形态多为梭形、多角形,倍增时间约为38h。ALP染色、Ⅰ型胶原免疫组化染色及Von Kossa钙染色阳性,证实所培养的细胞为成骨细胞。比较传代培养第6天时细胞数,MM患者成骨细胞数目(5.77±1.91)较正常对照组(7.55±2.54)减少(p<0.05)。培养4周后行Von-Kossa’s染色,骨髓瘤患者和正常对照组的成骨细胞均可见矿化物沉积,但是骨髓瘤患者(6.12±1.63)明显少于正常对照组(15.83±2.23)(p<0.05)。2.正常对照组加入MM血清共培养后,比较培养传代第6天时成骨细胞增殖数目(7.38±1.13)较正常培养组(8.25±2.59)减少(p<0.05),硼替佐米对正常人的成骨细胞没有明显的促进或抑制作用(p>0.05)。在MM组中比较培养传代并加入硼替佐米后第6天时细胞数,MM患者硼替佐米组成骨细胞数目(8.94±2.09)较正常培养组(6.33±1.51)明显增多(p<0.05),MM血清组成骨细胞数目与正常培养组比较无统计学意义(p>0.05)。成骨细胞传代培养4周后,行Von Kossa’s钙染色,可见MM患者加入硼替佐米培养组(8.83±1.94)的钙质沉积多于MM正常培养组(6.12±1.63)(p<0.05),但仍少于正常对照组(15.83±2.23)(p<0.05)。流式细胞仪检测各组成骨细胞均不表达CD138、CD45、CD34。MM血清中IL-7水平(2.07±0.71)高于正常对照组(1.62±0.15)(p<0.05)。MM正常培养组、MM硼替佐米组及正常对照组均可表达BMP2 mRNA, MM正常培养组BMP2 mRNA无明显表达。结论：使用骨髓培养法,可于体外培养MM患者成骨细胞。MM患者成骨细胞的增殖分化及成骨能力较正常人明显减弱。硼替佐米是一种成骨细胞正向调控因子,促进成骨细胞增殖分化及成骨能力；MM血清作为一种负向调控因子,可抑制成骨细胞的增殖分化。更多还原

【Abstract】 Objective:This study was to investigate the biological characteristics of osteoblasts cultured in vitro from bone marrow (BM) of multiple myeloma(MM) patients and to explore their generation and osteogenic potential. Then the affects by some factors such as bortezomib and MM patient serum on the osteogenic potential of osteoblasts from MM patients and normal subjects were observed.Methods:Fifteen MM patients and 10 healthy donors as controls were enrolled in this study. Osteoblasts from BM of MM patients were cultured in vitro. The morphology of these cells were observed, the growth pattern and double time were detected. The cultured cells were identified by ALP staining,Von Kossa staining and type I collagen immunohistochemistry assay. The generation and osteogenic potential of osteoblasts from MM patients and normal subjects were compared. The changes of their osteogenic potential and biological characteristics were observed. The antigens (CD34、CD138、CD45) on osteoblasts were examined with flow cytometric assay.The levels of IL-7 were measured with ELISA. The BMP2 mRNAs were measured by RT-PCR.Results:1. Osteoblasts from BM of MM patients could be cultured in vitro with dexamethasone,β-sodium glycerophosphate and vitamin C. The shape of the cells was almost spindle and polygon.Their double time was about 38 hours. ALP staining,Von Kossa staining and type I collagen immunohistochemistry staining were all positive to verify the osteoblasts. The quantity of osteoblasts from MM patients (5.77±1.91) was less than normal subjects (7.55±2.54) at the 6th day culture(p<0.05).There were calcium depositions in both groups after cultured for 4weeks culture. But the depositions of MM patients (6.12±1.63) was less than that of normal controls (15.83±2.23) (p＜O.05)2. The osteoblasts cultured with MM patient serum (7.38±1.13) were less than those without patient serum (8.25±2.59) (p<0.05). Bortezomib could not affect the cultured cells from normal person (p>0.05), but increased those from MM patients after 6days culter (8.94±2.09 vs 6.33±1.51) (p<0.05). After 4weeks culture, Von Kossa staining showed that there was more calcium depositions in MM ostesblasts with bortezomib (8.83±1.94) than those without bortezomib (6.12±1.63) (p<0.05), but less than those from normal controls. CD138, CD45, CD34 were not detected on the cultured cells. The level of IL-7 in serum of MM patients(2.07±0.71) was higher than that of normal controls(1.62±0.15)(p<0.05). The expression of BMP2 mRNA was seen in the normal osteoblasts and MM osteoblasts culured with bortezomib, but not seen in those without bortezomib.ConclusionsOsteoblasts could be cultured in vitro from BM of MM patients. The proliferation and osteogenic potential of osteoblasts from MM patients were decreased. Bortezomib was a positive regulatory of osteoblasts and MM patient serum was a negtive. They both could affect the proliferation and osteogenic potential of osteoblasts.更多还原