唑来膦酸诱导骨巨细胞瘤基质细胞向成骨细胞分化的实验研究

Zoledronate inducing differentiation of giant cell tumor of bone stromal cell into osteoblasts

目的探讨唑来膦酸诱导骨巨细胞瘤基质细胞向成骨细胞分化的可行性。方法取我院收治的7例唑来膦酸治疗前后的患者肿瘤组织切片行HE染色、茜素红染色、碱性磷酸酶(ALP)染色以及成骨前因子Ⅰ型胶原和骨唾液蛋白(BSP)的免疫组化染色。取10例未予唑来膦酸治疗的患者术中切除的骨巨细胞瘤新鲜组织进行原代培养,待骨巨细胞瘤基质细胞贴壁生长后分别给予1μmol/L唑来膦酸诱导12天后行ALP染色、免疫组化法检测Ⅰ型胶原和RT-PCR检测Cbfa-1的表达。结果 (1)唑来膦酸治疗前,肿瘤组织切片中骨巨细胞瘤基质细胞BSP染色阴性或低至中度阳性、Ⅰ型胶原染色阴性或轻度阳性,茜素红和ALP染色均为阴性;治疗后,茜素红染色阳性,ALP染色弱阳性,BSP和Ⅰ型胶原染色均转为强阳性,3例患者组织切片中HE染色发现类骨质或骨矿化的增加。(2)原代培养的骨巨细胞瘤基质细胞中予1μmol/L唑来膦酸诱导12天,空白对照组ALP和Ⅰ型胶原染色均为弱阳性表达,唑来膦酸诱导组呈阳性表达。RT-PCR检测发现唑来膦酸诱导组骨巨细胞癌基质细胞中Cbfa-1表达,而空白对照组未见Cbfa-1表达。结论体内外研究初步证实,骨巨细胞瘤基质细胞具有向成骨细胞分化的潜能并在唑来膦酸的诱导下向成骨细胞分化,这从细胞和分子生物学水平部分解释了临床上长期应用唑来膦酸治疗后肿瘤病灶和术后残留囊壁中的明显骨生成的现象。

Objective To explore the feasibility of zoledronate inducing differentiation of giant cell tumor of bone（GCT） stromal cell into osteoblasts.Methods In this study,the feasibility of zoledronate inducing osteoblastic differentiation of GCT stromal cell was explored with cell culture by microscopy,immunohistochemistry and RT-PCR methods.By HE-staining,alkaline phosphatase（ALP） and alizarin red staining,the expression of several osteoblast differentiatin-specific markers collagen type Ⅰ and bone sialoprotein（BSP） using immunohistochemistry in specimens obtained at the time of biopsy and those obtained following resection;GCT primary cultures from 10 un-treated patients were established.GCT stromal cells in cultures obtained after the 5th passage were studied for regulation of expression of collagen type Ⅰ,ALP activity by zoledronate.Collagen type Ⅰ was assayed using immunohistochemistry and ALP staining in GCT stromal cells was cultured for 12 days in the presence（treatment group） or absence（control group） of zoledronate in 1μmol/L concentrations.Results Among the cases in control group,majority of stromal tumour cells were found weakly or moderately positive in BSP and negative or weakly positive in collagen type Ⅰ.The majority of osteoclast-like giant cells did not stain for collagen type Ⅰ.Osteoclast-like giant cells were moderately positive for BSP.However,in treatment group,the stromal cells and osteoclast-like giant cells were all strongly positive in BSP and collagen Ⅰ.The histological sections（HE staining,alizarin red staining and ALP staining） in control group were compared with those in treatment group in order to evaluate the response to zoledronate.Topical increase in osteoid and mineralized bone was observed in some treatment cases,typically in sections taken at the margins of the lesion.Among the majority of zoledronate treatment cases,alizarinred staining was strongly and ALP staining was moderately positive while two types of stainings were negative in control group.ALP and collagen Ⅰ staining was minimal in GCT stromal cells in control cultures.Treatment with zoledronate markedly increased the intensity and extent of ALP and collagen Ⅰ staining.By using RT-PCR,the mRNA expression of Cbfa-1 was evaluated,the key transcription factor that was specifically activated during osteoblast differentiation in GCT stromal cells.The Cbfa-1 was hardly detected in GCT stromal cells in control cultures.Following zoledronate treatment,the PCR analysis show that Cbfa-1 mRNA was present in GCT stromal cells.Conclusion GCT stromal cells may have an osteoblastic lineage and retain the ability to differentiate into osteoblast.It may be confirmed the stimulation of osteogenic differentiation in GCT stromal cells by zoledronate.