重组嗜水气单胞菌Aeromonas hydrophila 4AK4中3-羟基丁酸3-羟基己酸共聚酯PHBHHx的发酵生产

3-羟基丁酸和3-羟基己酸共聚酯（PHBHHx）是一种性能优良的新型生物可降解材料,其机械和加工性能与3-羟基己酸（3HHx）在共聚物中的含量密切相关。在嗜水气单孢菌Aeromonas hydrophila 4AK4中引入了编码β酮基硫解酶 (β-ketothiolase)的phbA基因和编码乙酰乙酰辅酶A还原酶(AcetoacetylCoA reductase)的phbB基因,使重组菌增加了一条利用乙酰辅酶A合成3羟基丁酸-CoA的代谢途径,这使得利用非相关性碳源调控PHBHHx的单体组成比例成为可能。利用葡萄糖酸钠和月桂酸作为碳源,对重组Aeromonas hydrophila 4AK4进行了摇瓶培养及5 L发酵罐培养的研究。在摇瓶实验中,通过改变碳源中两种组分的比例,可以使A. hydrophila 4AK4合成的PHBHHx中的3HHx摩尔含量由原来的15%左右降低到3%～12 %,成功地实现了对PHBHHx单体组成的调控;当以月桂酸为唯一碳源时,在5 L发酵罐中,经过56 h的培养,获得了51.5 g/L的细胞干重（CDW）,其中62 %为PHBHHx,3HHx在PHBHHx中的摩尔含量为9.7 %;当以1:1的葡萄糖酸钠和月桂酸为碳源时,48 h的5 L发酵罐培养获得了32.8 g/L的CDW和52 %的PHBHHx含量,其中3HHx在PHBHHx中的摩尔含量为6.7 %。结果证明了该重组菌在大规模生产单体组成可控PHBHHx方面具有很大的应用潜力。     

重组嗜水气单胞菌Aeromonas hydrophila 4AK4中3-羟基丁酸3-羟基己酸共聚酯PHBHHx的发酵生产

3-羟基丁酸和3-羟基己酸共聚酯(PHBHHx)是一种性能优良的新型生物可降解材料,其机械和加工性能与3-羟基己酸(3HHx)在共聚物中的含量密切相关。在嗜水气单孢菌Aeromonas hydrophila 4AK4中引入了编码β-酮基硫解酶(β-ketothiolase)的phbA基因和编码乙酰乙酰辅酶A还原酶(Acetoacetyl-CoA reductase)的phbB基因,使重组菌增加了一条利用乙酰辅酶A合成3-羟基丁酸-CoA的代谢途径,这使得利用非相关性碳源调控PHBHHx的单体组成比例成为可能。利用葡萄糖酸钠和月桂酸作为碳源,对重组Aeromonas hydrophila 4AK4进行了摇瓶培养及5L发酵罐培养的研究。在摇瓶实验中,通过改变碳源中两种组分的比例,可以使A,hydrophila 4AK4合成的PHBHHx中的3HHx摩尔含量由原来的15％左右降低到3％～12％,成功地实现了对PHBHHx单体组成的调控;当以月桂酸为唯一碳源时,在5L发酵罐中,经过56h的培养,获得了51．5g／L的细胞干重(CDW),其中62％为PHBHHx,3HHx在PHBHHx中的摩尔含量为9．7％;当以1：1的葡萄糖酸钠和月桂酸为碳源时,48h的5L发酵罐培养获得了32．8g／L的CDW和52％的PHBHHx含量,其中3HHx在PHBHHx中的摩尔含量为6．7％。结果证明了该重组菌在大规模生产单体组成可控PHBHHx方面具有很大的应用潜力。     

嗜水气单胞菌WQ中PHBHHx的合成及其分子基础研究

聚羟基脂肪酸酯(Polyhydroxyalkanoate,PHA)是一系列生物合成的高分子材料,其单体可由多种3-羟基脂肪酸(3-hydroxyalkanoate,3HA)构成^[1]。PHA物理和机械性能的变化很大,从高脆性到弹性体,这跟它们的单体成分有很大关系^[2]。短链和中长链单体共聚的PHA比短链单体或中长链单体聚合得到的PHA有着更好的性能^[3]。在1994年,豚鼠气单胞菌(Aeromonas caviae)FA440被发现能以偶数碳原子数脂肪酸或植物油作为碳源在体内积累PHBHHx^[4]其PHA生物合成基因被成功克隆^[5]。根据亚基数目和底物特异性,PHA合成的关键酶,即PHA合酶或PhaC,被分成了3种类型。A．caviae的PHA合酶属于第1类PHA合酶^[6]。PHA合酶的一些类型含有一些保守的基因序列,该特征可被用于克隆,特别是第Ⅱ类PHA合酶^[2,8]。嗜水气单胞菌(Aeromonas hydrophila)WQ和A．hydrophila 4AK4是能够合成PHBHHx的另外两种菌株,其中A．hydrophila 4AK4已被用作大规模生产PHBHHx。就目前来说,不管生长条件怎么改变,其合成的PHBHHx中3羟基己酸单体(3-hydroxyhexanoate,3HHx)的含量始终在12％～17％之间变化^[9]。而A．hydrophila WQ合成的PHBHHx中则含有6％～14％ 3HHx。本论文研究了A．hydrophila WQ的PHA生物合成及其分子基础。     

PhaP介导EGFR靶向多肽修饰的肿瘤靶向PHBHHx纳米药物递送载体的构建

聚羟基脂肪酸(PHA)颗粒表面结合蛋白Pha P具有与疏水性高分子材料表面紧密结合的能力,本研究将EGFR靶向多肽(ETP)与PhaP进行融合表达,构建了ETP-PhaP融合蛋白表达的重组工程菌Escherichia coli BL21(DE3)(pPI-ETP-P)。经对工程菌株的诱导表达及ETP-PhaP融合蛋白的纯化后,通过PhaP蛋白介导能够有效地将ETP-PhaP融合蛋白修饰于3-羟基丁酸-3-羟基己酸共聚酯(PHBHHx)纳米微球表面,构建成为具有EGFR靶向作用的药物递送载体。分别检测宫颈癌细胞系SiHa(EGFR高表达)和CaSKi(EGFR低表达)对ETP-PhaP修饰的PHBHHx纳米药物载体和未经修饰的纳米药物载体的吞噬情况。结果显示,纯化的ETP-PhaP融合蛋白能够很好地吸附于PHBHHx颗粒的表面,经ETP-PhaP融合蛋白修饰的PHBHHx纳米药物载体对EGFR高表达的宫颈癌Si Ha细胞的靶向效果强于EGFR低表达的CaSKi细胞系。这一结果表明了PhaP介导的PHBHHx纳米微球表面EGFR靶向多肽修饰具有简便、高效的优势,为疏水性纳米药物载体表面功能多肽修饰提供了一种新策略。     

寡聚羟基丁酸酯OPHB及其衍生物的细胞代谢活性研究

羟基丁酸和羟基己酸共聚酯（poly(hydroxybutyrate-co-hydroxyhexanoate),PHBHHx）以其良好的生物相容性而具有广泛的生物医学应用前景。其降解产物之一寡聚羟基丁酸酯（oligo(polyhydroxubutyrate),OPHB）,广泛存在于自然界的生物体中,并承担各种功能。采用甲醇降解法制备OPHB的甲酯衍生物,MTT实验研究OPHB及其甲酯衍生物对人微血管内皮细胞（human mammary epithelial cells,HMECs）代谢活性的作用。研究结果表明,OPHB对HMECs细胞活性有20%的促进作用,在半饥饿培养条件下细胞活性提高了80%。而制备的OPHB甲酯衍生物有一定的细胞毒性细胞代谢活性降低50%。     

3-羟基丁酸与3-羟基己酸共聚酯在心血管组织工程中的应用

为了研究可降解聚合材料3-羟基丁酸与3-羟基己酸共聚酯 (3-hydroxybutyrate-co-3-hydroxyhexanoate, PHBHHx)的血管内生物相容性, 采用脱细胞羊肺动脉为支架, 以PHBHHx涂层, 构建复合补片(Hybrid patch), 植入New Zealand兔腹主动脉内(12只), 以脱细胞未涂层羊肺动脉片(Uncoated patch)做为对照(12只)。分别于术后第1、4和12周处死动物, 取出移植补片进行组织学、免疫荧光染色、扫描电镜和钙含量测定。结果表明: hybrid patch管腔面光滑无血栓, 内膜增生适度, 再细胞化完全; 免疫荧光染色检测, 新生内膜组织中类内皮细胞呈CD31阳性反应, 单层连续排列, 间质细胞呈现SMA阳性反应; 钙含量测定, hybrid patch明显低于uncoated patch(P<0.05)。由此认为: PHBHHx的血管内生物相容性满意, 是心血管组织工程较为理想的腔内涂层材料。     

3-羟基丁酸与3-羟基己酸共聚酯在心血管组织工程中的应用

为了研究可降解聚合材料3-羟基丁酸与3-羟基己酸共聚酯 (3-hydroxybutyrate-co-3-hydroxyhexanoate, PHBHHx)的血管内生物相容性, 采用脱细胞羊肺动脉为支架, 以PHBHHx涂层, 构建复合补片(Hybrid patch), 植入New Zealand兔腹主动脉内(12只), 以脱细胞未涂层羊肺动脉片(Uncoated patch)做为对照(12只)。分别于术后第1、4和12周处死动物, 取出移植补片进行组织学、免疫荧光染色、扫描电镜和钙含量测定。结果表明: hybrid patch管腔面光滑无血栓, 内膜增生适度, 再细胞化完全; 免疫荧光染色检测, 新生内膜组织中类内皮细胞呈CD31阳性反应, 单层连续排列, 间质细胞呈现SMA阳性反应; 钙含量测定, hybrid patch明显低于uncoated patch(P<0.05)。由此认为: PHBHHx的血管内生物相容性满意, 是心血管组织工程较为理想的腔内涂层材料。     

代谢工程技术调控3-羟基丁酸与3-羟基己酸共聚酯PHBHHx的微生物合成

3-羟基丁酸和3-羟基己酸共聚酯(PHBHHx)是一种新型生物可降解材料,其性能与3-羟基己酸(3HHx)在共聚物中的摩尔百分含量密切相关。本研究在两株嗜水性气单孢菌Aeromonas hydrophila WQ和Aeromonas hydrophila 4AK4中分别引入了编码酯酰辅酶A脱氢酶的yafH基因和编码合成3-羟基丁酸-CoA的phbA和phbB基因,将A.hydrophila WQ合成的PHBHHx中的3HHx的摩尔含量由3％—5％提高到20％以上;而A.hydrophila 4AK4合成的PHBHHx中的3HHx摩尔含量则由15％左右降低到3％-12％。成功地实现了对PHBHHx单体组成的调控。     

通过研究改性壳聚糖与细胞的相互作用评价其生物相容性

利用细胞生物学的方法, 研究了四种不同的细胞在经过改性的壳聚糖（ＣＨＩＴＯＳＡＮ） 膜上的生长,测定了细胞相对黏附力、细胞初始黏附率, 并利用ＦＤＡ 实验测定了细胞活力,从而从多个方面评价了这几种不同材料的生物相容性。实验结果表明,与明胶交联的壳聚糖膜明显比其它两种膜有利于细胞的黏附和生长,为进一步对材料进行筛选奠定了基础。     

膜性高分子材料在兔眼部的生物相容性

目的评价聚羟基脂肪酸(polyhydroxyalkanoates,PHA)、聚乳酸(polylacetic acid,PLA)和聚己内酯(polycaprolactone,PCL)三种膜性高分子材料在兔眼部的生物相容性。方法将24只新西兰兔随机分为4组,每组6只。PHA、PLA、PCL为实验组,材料植入兔右眼结膜下。假手术组结膜下钝性分离,但不植入任何高分子材料。使用裂隙灯显微镜观察并记录植入后不同时间手术眼的反应并评分。裂隙灯下观察材料的吸收时间。术后4周和16周取眼球,行HE染色、Masson染色和天狼猩红染色分别定性观察组织结构和炎症细胞、胶原纤维和胶原纤维的亚型与排列方向。结果术眼刺激性评分等级各组均不高于"轻度刺激性"。结膜下吸收时间PHA、PLA和PCL组分别是16周,12周和大于16周。组织学观察术后4周PHA、PLA和PCL组均形成材料包裹囊腔,囊壁以纤维组织为主,伴有毛细血管形成和炎性细胞浸润,以中性粒细胞为主。胶原纤维染色与假手术组无明显差异,以Ⅰ型和Ⅲ型为主,大致呈平行排列。术后16周PHA和PLA组材料已不可查及,包裹囊腔结构不规则,而PCL材料整体可查及,包裹囊腔规则。各组未见毛细血管,偶见淋巴细胞浸润。胶原纤维与假手术组无明显差异,以Ⅰ型和Ⅲ型为主,仍大致呈平行排列。结论 PHA、PLA和PCL三种膜性高分子材料在兔眼具有较好的生物相容性,结膜下吸收时间分别是16周,12周和大于16周。     

Effects of chitosan/collagen substrates on the behavior of rat neural stem cells

Spinal cord and brain injuries usually lead to cavity formation. The transplantation by combining stem cells and tissue engineering scaffolds has the potential to fill the cavities and replace the lost neural cells. Both chitosan and collagen have their unique characteristics. In this study, the effects of chitosan and collagen on the behavior of rat neural stem cells (at the neurosphere level) were tested in vitro in terms of cytotoxicity and supporting ability for stem cell survival, proliferation and differentiation. Under the serum-free condition, both chitosan membranes and collagen gels had low cytotoxicity to neurospheres. That is, cells migrated from neurospheres, and processes extended out from these neurospheres and the differentiated cells. Compared with the above two materials, chitosan-collagen membranes were more suitable for the co-culture with rat neural stem cells, because, except for low cytotoxicity and supporting ability for the cell survival, in this group, a large number of cells were observed to migrate out from neurospheres, and the differentiating percentage from neurospheres into neurons was significantly increased. Further modification of chitosan-collagen membranes may shed light on in vivo nerve regeneration by transplanting neural stem cells.     

小鼠胚胎干细胞结合丝素材料向神经细胞分化的实验研究

以小鼠胚胎干细胞（ES）为种子细胞,使用改良的4-/4+ RA方案,诱导小鼠ES细胞在丝素材料上向神经细胞分化,探讨丝素材料对其生长、黏附、分化等情况的影响。将小鼠ES细胞悬浮培养4 d得到的拟胚体（EBs）分别接种到经丝素膜和明胶包被的培养皿上进行诱导,比较不同材料上EBs的贴壁率及向神经元分化的比率。结果表明EBs在明胶和柞蚕丝素蛋白膜（TSF）上贴壁较快,平均贴壁率为90.3%和84.4%,在桑蚕丝素蛋白膜（SF）上贴壁较慢,贴壁率低,仅为38.5%,同时三者神经元的分化比率均能达到40%以上,无明显差异。通过以上实验,我们得出,TSF有望成为小鼠ES细胞向神经细胞分化的支架材料。     

Peripheral sensory neurons differentiate from neural precursors derived from human embryonic stem cells

Abstract Neural precursors have been derived from human embryonic stem cells (hESC) using the bone morphogenetic protein antagonist noggin. These neural precursors can be further differentiated to produce neural cells that express central nervous system (CNS) markers. We have recently shown that naïve hESC can be directed to differentiate into peripheral sensory (PS) neuron-like cells and putative neural crest precursors by co-culturing with PA6 stromal cells. In the present study, we examine whether hESC-derived neural precursors (NPC) can differentiate into the peripheral nervous system, as well as CNS cells. As little as 1 week after co-culture with PA6 cells, cells with the molecular characteristics of PS neurons and neural crest are observed in the cultures. With increased time in culture, more PS-like neurons appear, in parallel with a reduction in the neural crest-like cells. These results provide the first evidence that neural precursors derived from hESC have the potential to develop into PS neurons-like as well as CNS-like neuronal cells. About 10% of the cells in NPC-PA6 co-cultures express PS neuron markers after 3 weeks, compared with <1% of hESC cultured on PA6. This enrichment for peripheral neurons makes this an attractive system for generation of peripheral neurons for pathophysiology study and drug development for diseases of the peripheral nervous system such as Familial Dysautonomia and varicella virus infection.     

Human neural progenitor cells derived from embryonic stem cells in feeder-free cultures

Derivation of human neural progenitors (hNP) from human embryonic stem (hES) cells in culture has been reported with the use of feeder cells or conditioned media. This introduces undefined components into the system, limiting the ability to precisely investigate the requirement for factors that control the process. Also, the use of feeder cells of non-human origin introduces the potential for zoonotic transmission, limiting its clinical usefulness. Here we report a feeder-free system to produce hNP from hES cells and test the effects of various media components involved in the process. Five protocols using defined media components were compared for efficiency of hNP generation. Based on this analysis, we discuss the role of basic fibroblast growth factor (FGF2), N2 supplement, non-essential amino acids (NEAA), and knock-out serum replacement (KSR) on the process of hNP generation. All protocols led to down-regulation of Oct4/POU5F1 expression (from 90.5% to <3%), and up-regulation of neural progenitor markers to varying degrees. Media with N2 but not KSR and NEAA produced cultures with significantly higher (p<0.05) expression of the neural progenitor marker Musashi 1 (MSI1). Approximately 89% of these cells were Nestin (NES)+ after 3 weeks, but they did not proliferate. In contrast, differentiation media supplemented with KSR and NEAA produced fewer NES+ (75%) cells, but these cells were proliferative, and by five passages the culture consisted of >97% NES+ cells. This suggests that KSR and NEAA supplements did not enhance early differentiation but did promote proliferating of hNP cell cultures. This resulted in an efficient, robust, repeatable differentiation system suitable for generating large populations of hNP cells. This will facilitate further study of molecular and biochemical mechanisms in early human neural differentiation and potentially produce uniform neuronal cells for therapeutic uses without concern of zoonotic transmission from feeder layers.     

神经干细胞向少突胶质前体细胞的定向分化诱导

本研究采用神经胶质瘤细胞株(B104 neuroblatoma cells,B104 cells)培养上清(B104CM)和碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF),将冷冻复苏的大鼠胚胎脊髓神经干细胞(neural stem cells,NSCs)定向诱导为少突胶质前体细胞(oligodendrocyte precusor cells,OPCs)。形态学和免疫组化的结果显示,诱导后95％以上的细胞具有双极或多极突起的典型OPCs形态,并表达A285和血小板源生长因子受体-α(platelet derived growth factor receptor-α,PDGFR-α等0PCs标志,所有PDGFR-α阳性的OPCs均不表达β-Tublin Ⅲ,其中仅少量细胞表达胶质原纤维酸性蛋白(glia fibrillary acidic protein,GFAP)。在B104CM和bFGF共存的培养条件下,悬浮培养的OPCs可大量增殖形成少突胶质细胞球,该细胞球可通过传代继续扩增,且扩增的OPCs仍能维持其特有的形态和自我增殖的特性。撤去bFGF和B104CM后,OPCs能进一步分化为成熟的少突胶质细胞(oligodendrocytes,OLs)或Ⅱ型星形胶质细胞。实验表明,诱导NSCs产生的OPCs在形态、增殖以及分化格局等方面均与已报道的存在于胚胎脑区的O-2A前体细胞相类似。该培养系统可为实验性细胞移植的研究提供丰富的细胞来源。     

Genetic approach to track neural cell fate decisions using human embryonic stem cells

With their capability to undergo unlimited self-renewal and to differentiate into all cell types in the body, human embryonic stem cells (hESCs) hold great promise in human cell therapy. However, there are limited tools for easily identifying and isolating live hESC-derived cells. To track hESC-derived neural progenitor cells (NPCs), we applied homologous recombination to knock-in the mCherry gene into the Nestin locus of hESCs. This facilitated the genetic labeling of Nestin positive neural progenitor cells with mCherry. Our reporter system enables the visualization of neural induction from hESCs both in vitro (embryoid bodies) and in vivo (teratomas). This system also permits the identification of different neural subpopulations based on the intensity of our fluorescent reporter. In this context, a high level of mCherry expression showed enrichment for neural progenitors, while lower mCherry corresponded with more committed neural states. Combination of mCherry high expression with cell surface antigen staining enabled further enrichment of hESC-derived NPCs. These mCherry⁺NPCs could be expanded in culture and their differentiation resulted in a down-regulation of mCherry consistent with the loss of Nestin expression. Therefore, we have developed a fluorescent reporter system that can be used to trace neural differentiation events of hESCs.     

Isolation of neural crest-derived stem cells from rat embryonic mandibular processes

BACKGROUND INFORMATION: Substantial evidence indicates the existence of NCSCs (neural crest-derived stem cells) in embryonic mandibular processes; however, they have not been fully investigated or isolated. The aim of the present study was to isolate stem cells from mandibular process during embryonic development by MACS (magnetic-activated cell sorting). The findings show that the cells are multipotent and self-renewing. RESULTS: LNGFR (low-affinity nerve-growth-factor receptor)+ cells were isolated from rat embryonic mandibular processes by MACS. The cells were grown in clonal culture by limiting dilution to assess their developmental potential. Clone analysis indicated that, first, LNGFR+ cells are multipotent, being able to generate at least neurons and Schwann cells, similar to peripheral neural crest stem cells. Secondly, multipotent LNGFR+ cells generate multipotent progenies, indicating that they are capable of self-renewal and therefore are stem cells. Thirdly, manipulation of the medium supplementation alters the fate of the isolated LNGFR+ cells. CONCLUSIONS: These results indicate that LNGFR antibodies label NCSCs with high specificity and purity, and suggest that positive selection using these antibodies may become the method of choice for obtaining multipotent cells from rat embryonic mandibular processes for tissue engineering or regenerative therapeutic use.     

神经干细胞研究进展

神经干细胞研究是当今生命科学研究的热点之一。神经干细胞是神经系统发育过程中保留下来的具有自我更新和多分化潜能的原始细胞。随着对神经干细胞认识的不断深入,其临床应用前景与价值得到了越来越多研究者的肯定。从神经干细胞的生物学特征、来源、培养鉴定、分化及应用等几个方面对目前的研究做一概述。