课题背景：课题受国家自然科学基金和卫生部部属临床重点学科项目资助，在之前的研究中已顺利构建转化生长因子β2的真核表达载体，并在转染人关节软骨细胞和维持其表型的研究中获得成功。

偏倚或不足：①虽然在转染后48 h和4周均检测到目的基因的表达，但基因整合和表达的长期稳定性尚需进一步确定，采用病毒载体进行转化实验或许可以提高基因整合率和表达稳定性。②骨髓间质前体细胞在体外单层培养中可诱导表达软骨相关基因和蛋白，但尚不能证实其能分化为成熟软骨细胞，尤其不能确定其具有关节软骨的生物学特性，下一步可利用生物反应器或者进行体内实验深入分析。

术语解析：基因转染是将具生物功能的核酸（RNA或DNA）转移或运送到细胞内，并使核酸在细胞内维持其生物功能的核酸转移技术。在组织工程研究中，靶细胞不仅是作为目的基因的携带者，而且还是组织修复的细胞成分。基因转染的方法包括磷酸钙共沉淀法、电穿孔法、机械法以及脂质体法等，其中脂质体转染的转染效率相对较高，操作简便，是实验常用的转染方法。

摘要
目的：人脐血造血干细胞得以移植于小鼠体内，其理论基础是放射线照射使小鼠骨髓枯竭，龛位腾出，为移植的脐血造血干细胞提供空间，而猪苓多糖是从中药猪苓中提取的多糖成分，具有免疫调节抗肿瘤作用，也是一种非T细胞促有丝分裂素，对放射线照射有一定的防护作用。观察猪苓多糖对脐血造血干细胞体外扩增及干细胞移植后免疫重建效应。
方法： 实验于2004-06/2006-05在兰州大学基础医学院免疫研究所完成。①实验材料：清洁级BALB/c小鼠由兰州生物制品研究所提供，8周龄，体质量约20 g，雌雄各半，实验过程中对动物处置符合动物伦理学标准。脐血样本由兰泰医院妇产科提供，产妇及家属对实验知情同意，并经医院伦理委员会批准。猪苓多糖由兰州大学第一医院提供。②实验方法：应用20，17.5，12.5，7.5 mg/L猪苓多糖体外扩增培养脐血造血干细胞，并测定细胞增殖率及CD34+细胞数。将BALB/c小鼠分为正常对照组：尾静脉、腹腔注射等量生理盐水；照射空白组：3.5Gy经60Coγ放射线照射联合尾静脉、腹腔注射等量生理盐水；照射药物组：3.5Gy经60Coγ放射线照射联合尾静脉注射等量生理盐水、腹腔注射猪苓多糖；照射移植组（n =15）：3.5Gy经60Coγ放射线照射联合脐血造血干细胞移植、腹腔注射等量生理盐水；照射移植药物组：3.5Gy经60Coγ放射线照射联合脐血造血干细胞移植、腹腔注射猪苓多糖。③实验评估：观察小鼠生存状况及血常规变化，流式细胞仪测定CD3、CD4、CD8、CD19水平。
结果：57只小鼠均进入结果分析。①12.5 mg/L猪苓多糖增殖效率最为显著（P < 0.01），流式细胞仪测定显示CD34+细胞数扩增了6.33倍。移植药物组小鼠死亡率最低，死亡高峰集中在照射移植后7~14 d左右。②移植药物组小鼠血象恢复正常，并且时间明显短于其他实验组（P < 0.05）。③细胞及体液免疫恢复情况，移植药物组小鼠各项指标水平与正常小鼠无差别（P > 0.05），与其他各组小鼠比较，除CD4、CD19水平与照射移植小鼠无差别外均有差别（P < 0.05）。
结论：猪苓多糖对脐血造血干细胞有明显扩增作用，并能促进脐血造血干细胞移植小鼠免疫造血重建。
关键词：猪苓多糖；脐血造血干细胞；体外扩增；动物模型；免疫重建

潘万龙，李淑萍，唐恩洁，赵粉琴，罗慧英，尹少甫.猪苓多糖对脐血造血干细胞体外扩增及干细胞移植后免疫重建的调节效应[J].中国组织工程研究与临床康复，2000，12(12):2216-2220
[www.zglckf.com/zglckf/ejournal/upfiles/08-12/12k-2216(ps).pdf]

Regulatory effects of polyporus umbellatus polyose on in vitro amplification of umbilical cord blood hematopoietic stem cells and immune reconstruction after umbilical cord blood hematopoietic stem cell transplantation

Abstract

AIM：Human umbilical cord blood hematopoietic stem cells can be transplanted into mice. It is based on vacation in mice bone marrow niches by irradiation. Polyporus umbellatus polyose drown from the Chinese medicine, polyporus umbellatus, has effects on immunoloregulation, tumor and irradiation prevention. This article observes immune reconstruction of polyporus umbellatus polyose on umbilical cord blood hematopoietic stem cell transplantation.

METHODS: Experiments were conducted at the Institute of Immunology, Basic Medical College, Lanzhou University from June in 2004 to May in 2006. ①Clear BALB/c mice, eight weeks old, weighing 20 g, of either sex, were supplied by Lanzhou Institute of Biological Products. Animal disposition met animal ethical standard. Cord blood was offered by the Lantai Hospital of Lanzhou. Parturiens and their family members signed the informed consent. The experimental procedures were approved by hospital ethics committee. Polyporus umbellatus polyose was provided by First Affiliated Hospital of Lanzhou University. ②20，17.5，12.5，7.5 mg/L polyporus umbellatus polyose was applied to culture umbilical cord blood hematopoietic stem cells in vitro. Cell proliferation rate and CD34+ cell count were measured. BALB/c mice were divided into 5 groups. Mice in the normal control group were treated with saline via caudal vein and abdominal cavity. Mice in the irradiation blank group were irradiated with 3.5Gy by 60Co gamma ray and treated with saline via caudal vein and abdominal cavity. Mice in the irradiation-drug group received 3.5Gy by 60Co gamma ray, saline via caudal vein and polyporus umbellatus polyose via abdominal cavity. Mice in the irradiation-transplantation group (n =15) were irradiated with 3.5Gy by 60Co gamma ray, treated with umbilical cord blood hematopoietic stem cell transplantation and saline via abdominal cavity. Mice in the irradiation-transplantation-drug group received 3.5Gy by 60Co gamma ray, umbilical cord blood hematopoietic stem cell transplantation and polyporus umbellatus polyose injection via abdominal cavity. ③Survival and changes in blood routine parameters of mice were examined. CD3, CD4, CD8 and CD19 levels were determined with a flow cytometer.

RESULTS: Totally 57 mice were involved in the result analysis. ①Proliferation efficiency was the highest in mice treated with 12.5 mg/L polyporus umbellatus polyose (P < 0.01). The results of the flow cytometer determination showed that number of CD34+ amplified 6.33 times. Death rate of mice was the lowest in the irradiation-transplantation-drug group, and a peak of death occurred at 7-14 days after the irradiation and transplantation. ②Hemogram recovered to normal in the irradiation-transplantation-drug group, and the time of recovery was shorter than that of other groups (P < 0.05). ③There was no significant difference in the recovery of cell and humoral immunity in mice of the irradiation-transplantation-drug group and normal mice (P > 0.05), so does the CD4 and CD19 levels compared with the irradiation-transplantation group. There were significant differences in other indexes (P < 0.05).

CONCLUSION: Polyporus umbellatus polyose can evidently amplify umbilical cord blood hematopoietic stem cells and promote immune and hematopoietic reconstruction in mice undergoing umbilical cord blood hematopoietic stem cell transplantation.

Pan WL, Li SP, Tang EJ, Zhao FQ, Luo HY, Yin SF.Regulatory effects of polyporus umbellatus polyose on in vitro amplification of umbilical cord blood hematopoietic stem cells and immune reconstruction after umbilical cord blood hematopoietic stem cell transplantation.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(12):2216-2220(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-12/12k-2216(ps).pdf]