【作者】 李丁；

【导师】 王小宁；

【作者基本信息】 华南理工大学 ， 微生物学， 2013， 博士

【摘要】 胚胎干细胞（ESC）能无限增殖，具有可以分化成各种细胞和组织的能力，是在组织工程和细胞治疗中理想的细胞资源。尽管在许多研究工作中，开发出了具有生物相容性的培养基质和适合细胞生长的生物反应器，用于体外干细胞的培养和维持，但是昂贵的细胞生长因子和多能性维持试剂依然使得对ESC进行大量生产是十分困难和具有挑战的。如今，传统中草药（TCHM）由于自身对疾病的治疗效果和对细胞毒性作用低等原因，开始得到了广泛关注。除了疾病治疗外，一些草药还可以通过激活淋巴细胞、增加自然杀伤细胞和抗氧化等方式提高机体免疫力，因此，TCHM也成为了拥有潜在促进ESC增殖能力的新型药物资源。本研究建立了一种简单、高效、可靠和高通量的筛选方法，用于评估TCHM提取物对细胞生长的作用效果。这一筛选平台建立在聚对苯二甲酸乙二酯（PET）为支架进行三维（3D）细胞培养的基础之上，选用转染后稳定表达绿色荧光蛋白（GFP）的小鼠ESC、人乳腺癌和结肠癌细胞系，并采用改良的384荧光孔板对细胞表达的荧光强度进行在线监测，从而对细胞生长动力学进行实时分析，实现动态反映药物对细胞生长影响的效果。通过检测不同浓度下脱氧胆酸、灵芝孢子粉（Gls）、银杏和淫羊藿的水提取物对细胞增殖和细胞毒性的作用，得到的检测结果与已有相关报道中的结果一致。在可靠性分析中，Z检验结果为0.5和1.0之间，证明这种细胞水平药物筛选（CBDS）平台提供的筛选结果变异性小，适合用于TCHM的高通量筛选（HTS）。Gls浓度为0.01%（w/v）时，mESC在旋转瓶中3D动态培养5d后扩增倍数为18.5，与没有添加药物的对照组（扩增13.6倍）相比，扩增倍数提高了36.1%，同时mESC对SSEA-1和Oct-4的表达维持在90%以上。分别将水溶性的灵芝多糖（PSS）和脂溶性的灵芝三萜（TTS）进行分离和提取，并在3D荧光CBDS系统中检测这两种有效成分对mESC细胞的影响，结果显示当PSS和TTS单独作用于mESC细胞时，没有增殖促进效果。对另外6种TCHM在ESC生长中的作用进行了考察，发现三七（Pn）、白术（Ram）、川芎（Rc）和Gls表现出促进ESC增殖的潜力。对这4种药物搭配成的11种药物组合继续进一步筛选，结果显示将Pn和Gls等量混合后，提取物（Pn/Gls）对ESC生长的促进效果十分明显，与没有药物处理的细胞相比，Pn/Gls在较低的剂量下（0.01%w/v）使ESC的比生长速率提高了29.5%（p <0.01），在较高剂量（0.1%w/v）时也使比生长速率增加了34.2%（p <0.05）。之后在旋转瓶反应器中对Pn/Gls在大规模培养环境中促进细胞生长的效果进行验证，5天培养结束后，添加0.01%Pn/Gls的培养基中生长的ESC扩增倍数达到了29.3，而对照组扩增倍数为16.8。此外，在Pn/Gls作用下生长的ESC，生长速率提高的同时对全能性基因阶段特异性胚胎抗原（SSEA-1）和Oct-4基因的表达水平并没有下降，而且细胞核型也没有受到破坏。因此，这种3D荧光细胞水平的筛选方法能够提供高效、可靠的方式，从TCHM中筛选ESC生长促进因子；而通过这种筛选体系得到的药物Pn/Gls在大规模生产高质量ESC的应用当中也具有很好的前景。更多还原

【Abstract】 The capability of unlimited proliferation as well as differentiation into all types of cellsand tissues make embryonic stem (ES) cells ideal cell sources for tissue engineering and celltherapy. Although several biocompatible matrices and bioreactors have been developed forculturing stem cells, mass production of ES cells is still challenging due to the high costs ofcell growth promoters and pluripotency-maintaining reagents. Nowadays, traditional Chineseherbal medicines (TCHM) are becoming popular because of their therapeutic effects and lowtoxicity. In addition to disease treatments, some herbal medicines can also enhance immunefunctions by activating the proliferation of lymphocytes, increasing the amount of naturalkiller (NK) cells, and scavenging free radicals to resist oxidation. Thus, TCHMs have thepotential to be a new source of growth promoters for stem cells, especially ES cells.In this work, a simple, reliable, high-throughput screening method was developed andused to assess the pharmaceutical effects of extracts of TCHM. This method is based on3-dimensional (3D) cultures in polyethylene terephthalate (PET) fibrous scaffolds of mouseembryonic stem (mES) and human colon cancer and breast cancer cells expressing enhancedgreen fluorescent protein (GFP) after transfection on modified384-well plates with onlinemonitoring of culture fluorescence for dynamic responses of cells to drugs present in culturemedia. Cell responses to deoxycholic acid and the extracts of3TCHM (Ganoderma lucidumspores, Ginkgo biloba, and Epimedium brevicornum) at various concentrations wereinvestigated for their effects on proliferation and cytotoxicity. The screening results wereconsistent with what have been reported in the literature, confirming the reliability of the newscreening approach. Reliability analysis indicated the value of Z factor is in the range of0.5and1.0, verifying the cell-based drug screening (CBDS) platform is feasible forhigh-throughput screening (HTS) of TCHM with low variation.In in vitro mass production test, mESC grew in spinner flask supplemented with0.01%(w/v) Gls for3D dynamic culture reached an18.5folds expansion after5d. Compared to theno drug treated control group (13.6folds), Gls increased the expansion fold of mESC by36.1%, and the fractions of cells expressing both SSEA-1and Oct-4remained at over90%.Two active ingredients, water-soluble polysaccharides (PSS) and lipid-soluble triterpenes(TTS), of Gls were separated and extracted to test the effects on mESC proliferation using3Dfluorescence CBDS platform. Little growth promotion effects were detected when PSS orTTS treated mESC individually. For further screening, the high-throughput cell-based method was applied for screeningTCHM for potential stem cell growth promoters. Mouse ES cells expressing enhanced GFPwere cultured in growth media supplemented with various TCHM extracts. Thedosage-dependent effects of TCHM extracts on cell growth, including proliferation andcytotoxicity, were assessed via GFP fluorescence measurement. Six more TCHM extractswere investigated, and among all the nine tested extracts Panax notoginseng (Pn), RhizomaAtractylodis macrocephalae (Ram), Rhizoma chuanxiong (Rc) and Ganoderma lucidumspores (Gls) showed potential to improve mES cell proliferation. Eleven mixtures of these4TCHMs were then studied, and the results showed that the mixture of Pn and Gls had thestrongest growth promoting effect, increasing the specific growth rate of mES cells by29.5%at a low dosage of0.01%(w/v) Pn/Gls (p <0.01) and34.2%at0.1%(w/v) Pn/Gls (p <0.05)compared to the control. The growth promoting effect of Pn/Gls was further confirmed withES cells cultured in spinner flasks. A29.3-fold increase in the total cell number was achievedin the medium supplemented with0.01%Pn/Gls after5days, while the control culture onlygave a16.8-fold increase. It is noted that mES cells cultured in Pn/Gls supplemented mediacan also preserve pluripotency and without damage on Karyotype.Therefore, the cell-based screening method can provide an efficient and high-throughputway to explore potential stem cell growth promoters from TCHMs, and Pn/Gls screened fromthis method is promising in large-scale production of high-quality ES cells.更多还原