为探索研制丙型肝炎疫苗的新途径，以期获得防治丙型肝炎的重组腺病毒减毒活疫苗，我们构建了表达丙型肝炎病毒（Hepatitis Cvirus, HCV）非结构蛋白3（non-structural protein3, NS3）抗原的重组腺病毒Rad-NS3，并检测其在体外表达。应用PCR从真核表达质粒pRC/NS3中扩增编码HCVNS3蛋白（329-935aa）的基因片段，定向克隆到重组腺病毒AdEasy-1系统的穿梭质粒pAdTrack-CMV上，采用细菌内同源重组”两步转化法”构建携带HCVNS3基因的重组腺病毒基因组质粒pAd-HCVNS3，转染293细胞，成功包装出重组腺病毒Rad-NS3，利用它有效地感染人肝癌细胞株HepG2，经RT-PCR及免疫印迹等不同方法检测表明，被感染细胞能表达HCVNS3蛋白，为后续进行重组腺病毒在动物体内诱导抗HCV免疫应答能力的研究奠定了基础。

探索在体外分离培养小鼠骨髓间充质干细胞（Bonemarrow mesenchy malstemcells, BMSCs）的最适条件。以密度梯度离心技术及贴壁筛选法相结合，在体外分离BALB/C小鼠的BMSCs，通过MTT法测定了不同离心力、不同换液时间、不同血清浓度、不同种植密度等因素对BMSCs分离和培养的影响。在500g×30min的离心力下分离细胞，加入10%的胎牛血清，原代按（12～20）×105/ml的细胞密度接种，接种24h后换液，继代种植密度为6.4～25.6×104/ml时最适宜细胞生长；在此条件下，细胞快速增殖，传代后8h贴壁达90%以上，24h便进入对数生长期，直至第5d，细胞约增殖3倍。本研究建立了在体外分离培养骨髓间充质干细胞的细胞生物学方法和技术参数。

探讨人胚胎成纤维细胞用于人胚胎干细胞体外长期培养的可行性，以含10%胎牛血清的DMEM（低糖）溶液为培养基，对人胚胎成纤维细胞的生长形态、对胰酶的敏感性、生长曲线及细胞周期进行研究，并与小鼠胚胎成纤维细胞作比较。结果显示，人胚胎成纤维细胞和小鼠胚胎成纤维细胞在体外均为贴壁生长型细胞，与小鼠胚胎成纤维细胞相比，人胚胎成纤维细胞生长更旺盛，且细胞寿命更长；在室温条件下，对0.255胰酶更敏感，消化时间不宜超过3mim。提示人胚胎成纤维细胞不仅在生长状况上与小鼠胚胎成纤维细胞类似，而且作为人胚胎干细胞体外长期培养的饲养层，在使用期限上优于后者，值得进一步研究。

METHODS: Kupffer cells were immunohistochemically stained by streptavadin-peroxidase conjugated method (S-P). The numbers of Kupffer cells in cancerous, para-cancerous and adjacent normal liver tissues of 48 HCCs were comparatively examined. RESULTS: The mean number of Kupffer cells in cancerous, para-cancerous and adjacent normal liver tissues was 12.7～6.8, 18.1～8.2 and 28.9～7.9 respectively. The number of Kuppfer cells in cancerous tissues was significantly lower than that in para-cancerous tissues (t=2.423, P<0.05) and adjacent normal liver tissues (t=2.521, P<0.05). As tumor size increased, the number of Kupffer cells in cancerous tissues significantly decreased (F=4.61, P<0.05). Moreover, there was also a significant difference in the number of Kupffer cells among well-differentiated, moderately-differentiated and poorly-differentiated cases(F=4.49, P<0.05). CONCLUSION: This study suggests that decrease of Kupffer cells in HCCs may play an important role in the carcinogenesis of HCQ the number of Kupffer cells in HCC is closely related to the size and differentiation grade of the tumor.