富血小板血浆对人骨髓间充质干细胞成骨分化的抑制效应

目的:一些理论质疑富血小板血浆对骨前体细胞成骨分化的作用,本实验拟验证富血小板血浆对体外培养的人骨髓间充质干细胞成骨分化的抑制效应。方法:实验于2005-05/11在南方医科大学组织工程试验室(省级)完成。①实验方法:抽取6名健康志愿者髂前上棘骨髓5mL进行体外细胞培养扩增,静脉血10mL以二次离心法制得富血小板血浆。诱导骨髓间充质干细胞时富血小板血浆与骨髓间充质干细胞均来自同一个体。②碱性磷酸酶染色:取第4代骨髓间充质干细胞,分为两组:富血小板血浆组加入富血小板血浆使终浓度为100g/L,单纯血清培养组仅加入等量胎牛血清。培养后第7天进行碱性磷酸酶染色,阳性细胞为胞质中呈现黑色颗粒或块状沉淀。③矿化结节染色:取第4代骨髓间充质干细胞,分组同上。培养后第19天以0.1%茜素红-TrisHcl(pH8.3)37℃下放置30min,矿盐沉积染色阳性为红色。④Cbfa1基因表达:取第4代骨髓间充质干细胞,分组同上。培养后第3,7,12,16天RT-PCR法检测骨髓间充质干细胞Cbfa1基因的表达。⑤形态学观察:实验过程中使用相差显微镜观察各组细胞生长情况及形态学变化。结果:①骨髓间充质干细胞碱性磷酸酶染色结果:培养后第7天,富血小板血浆组碱性磷酸酶阳性细胞数量较单纯血清培养组明显减少,且阳性细胞内灰黑色颗粒也明显减少,为弱阳性。②骨髓间充质干细胞矿化结节染色结果:培养后第19天,单纯血清培养组可见细胞表面有较多的矿盐沉积,但未形成明显的矿化结节。富血小板血浆组细胞表面只有稀少的矿盐沉积。③骨髓间充质干细胞cbfa1mRNA的表达:培养后第3,7,12,16天,随着培养时间的延长单纯血清培养组与富血小板血浆组cbfa1基因表达量均逐渐增高,同一时间点两组间cbfa1基因的表达基本相似。④骨髓间充质干细胞形态学变化:富血小板血浆组骨髓间充质干细胞增殖旺盛,细胞达到单层汇合的时间较单纯血清培养组明显缩短。单纯血清培养组细胞在完全汇合后开始出现聚合现象(14～16d),但趋向性不明显,未完全形成团簇;富血小板血浆组细胞在完全汇合后未出现聚合现象,细胞密集生长。培养初期两组细胞以梭形为主,多角形细胞较少,培养至14～16d单纯血清培养组多角形细胞较富血小板血浆组增多。结论:富血小板血浆可抑制人骨髓间充质干细胞碱性磷酸酶的分泌与矿盐沉积,对人骨髓间充质干细胞成骨分化的直接效应是抑制其分化。     

Comparison of the efficacy of intravesical Bacillus Calmette-Guérin with thiotepa, mitomycin C, poly I:C/poly-L-lysine and cis platinum in murine bladder cancer

The efficacy of intravesical Bacillus Calmette-Guérin for the treatment of the mouse bladder tumor MBT-2 was compared with that of thiotepa, mitomycin C, cis-diamminedichloroplatinum II and poly I:C/poly-L-lysine. MBT-2 cells were instilled into the bladder immediately after electrocauterization. Drug instillations were initiated 24 hours later and continued on a weekly basis for 4 weeks. Both Bacillus Calmette-Guérin and cis-diamminedichloroplatinum II significantly (p less than .0004) inhibited MBT-2 tumor implantation when compared to diluent-treated controls. Neither mitomycin C, thiotepa nor poly I:C/poly-L-lysine significantly inhibited tumor implantation. Mean tumor weights also were significantly (p less than .05) reduced in Bacillus Calmette-Guérin and cis-diamminedichloroplatinum II-treated mice, while tumor mean weights in mice treated with thiotepa, mitomycin C or poly I:C/poly-L-lysine were not significantly different than controls. These results suggest that the efficacy of intravesical Bacillus Calmette-Guérin in comparison with other drugs in the MBT-2 mouse bladder tumor model is similar to observations reported in human clinical trials in which intravesical Bacillus Calmette-Guérin was shown to be more effective than other cytotoxic drugs. These data further support the utility of the MBT-2 model for the study of the mechanisms by which Bacillus Calmette-Guérin inhibits bladder tumor growth.     

Serum ribonuclease in urologic cancer Relation to host immunocompetence

Increased circulating levels of S-RNase (serum ribonuclease), an enzyme with immunosuppressive properties, were demonstrated in 14 of 19 patients with prostatic carcinoma, 6 of 6 patients with renal cell carcinoma, 5 of 6 patients with bladder carcinoma, but only 1 of 8 patients with testis tumors. A significant correlation between S-RNase levels and the extent of tumor was demonstrated in patients with prostatic carcinoma, but not in patients with other urologic malignant conditions. Moreover, a significant correlation was demonstrated between S-RNase and host cell-mediated immunocompetence as measured by delayed cutaneous hypersensitivity to DNCB (dinitrochlorobenzene). The results suggest that the S-RNase assay may be of clinical use in experimental treatment programs to reflect fluctuations of tumor status when subtle effects on tumor growth may be obscured by the advanced tumor state.