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目的研究细胞表面黏附分子CD44对骨肉瘤细胞增殖、黏附和侵袭特性的影响,探讨骨肉瘤细胞生长侵袭的机制。方法流式细胞术和Westernblot检测3株骨肉瘤细胞系MG63、HOS和U2-OS中CD44的阳性表达率和相对蛋白含量;逆转录聚合酶链反应(RTPCR)法研究3株细胞系之间CD44mRNA的表达差异;同时应用MTT法、美蓝硼酸盐法和微孔迁移技术研究阻断CD44的作用前后3株细胞的增殖、黏附和侵袭能力的变化。结果HOS和U2-OS中CD44的阳性百分率均高于99%,但HOS中CD44的平均荧光强度显著高于U2-OS(P<0.01);而MG63中CD44阳性百分率仅为(2.10±0.46)%;Westernblot亦证明HOS中CD44的蛋白含量显著高于U2-OS(P<0.05),而MG-63中CD44表达为阴性;CD44mRNA在HOS和U2OS中的表达也显著高于MG-63(P值均<0.05)。HOS的侵袭性显著高于MG63和U2-OS(P值均<0.01);HOS与MG63的增殖速率和黏附性差异无统计学意义,但均显著高于U2OS(P值均<0.01)。应用中和抗体阻断CD44的作用之后,HOS和MG-63的黏附性均显著降低(P值均为0.03),U2-OS的黏附性无明显变化(P=0.93);HOS和U2-OS的侵袭力显著降低(P值均<0.01),而MG-63的侵袭力无明显改变(P=0.18);3株细胞的增殖速率均无显著变化(P值均>0.05)。结论CD44能促进骨肉瘤细胞系HOS的黏附和侵袭,并参与U2-OS的侵袭和MG-63的黏附过程,但是不影响骨肉瘤细胞的增殖速率。
Objective To study the effect of cell surface adhesion molecule CD44 on the proliferation, adhesion and invasion of osteosarcoma cells and to explore the mechanism of osteosarcoma cell growth and invasion. Methods The positive rate and relative protein content of CD44 in three osteosarcoma cell lines MG63, HOS and U2-OS were detected by flow cytometry and Western blotting. The expression of CD44 mRNA in three cell lines was analyzed by reverse transcription polymerase chain reaction (RT-PCR) . The MTT assay, methylene blue borate assay and micropore migration assay were used to investigate the proliferation, adhesion and invasion ability of the three cell lines before and after the blockade of CD44. Results The positive percentage of CD44 in HOS and U2-OS was higher than 99%, but the average fluorescence intensity of CD44 in HOS was significantly higher than that in U2-OS (P <0.01), while the positive percentage of CD44 in MG63 was only (2.10 ± 0.46) %; Western blot also showed that the protein content of CD44 in HOS was significantly higher than that in U2-OS (P <0.05), while the expression of CD44 in MG-63 was negative. The expression of CD44mRNA in HOS and U2OS was also significantly higher than that in MG-63 All <0.05). The invasiveness of HOS was significantly higher than that of MG63 and U2-OS (all P <0.01). There was no significant difference in the proliferation rate and adhesion between HOS and MG63, but both were significantly higher than U2OS (all P <0.01). The adhesion of HOS and MG-63 was significantly reduced by neutralizing antibodies against CD44 (P = 0.03), but there was no significant difference in the adhesion of U2-OS (P = 0.93). HOS and U2-OS (P <0.01), while the invasiveness of MG-63 did not change significantly (P = 0.18). There was no significant change in the proliferation rate of all the three cells (P> 0.05). Conclusion CD44 can promote the adhesion and invasion of HOS in osteosarcoma cell line, and participate in the invasion of U2-OS and the adhesion of MG-63, but it does not affect the proliferation rate of osteosarcoma cells.