微囊牛肾上腺髓质细胞的冻存及其活性评价

目的　建立一种微囊牛肾上腺髓质细胞 (bovinechromaffincell,BCC)的冻存方法 ,为临床推广微囊人工生物细胞技术移植异种细胞奠定基础。方法　微囊化牛肾上腺髓质细胞冻存以二甲基亚砜为保护剂 ,循序缓慢降温 (4℃ ,1h ,- 2 0℃ 2h ,- 5 0℃过夜 ,液氮 ) ,复苏时迅速将冻存管放入 37℃水浴中。通过检测其基础儿茶酚胺分泌量和在高钾 (5 8mmol/L)、乙酰胆碱 (10 -4mol/L)刺激下的分泌量来观察其功能活性。结果　冻存复苏后微囊BCC保留了儿茶酚胺的分泌功能 ,基础与刺激分泌量分别为 (3.2 0 7± 0 .35 0 )ng/ml和 (12 .4 96± 2 .30 2 )ng/ml,大约为冻存前微囊牛肾上腺髓质细胞分泌量的 80 % ;同时刺激后儿茶酚胺分泌增加 (P <0 .0 1)。结论　建立一种微囊牛肾上腺髓质细胞的冻存方法 ,通过功能检测证明该方法是有效和成功的     

移植用微囊化细胞低温保存的研究

目的：探索移植用微囊化细胞低温保存的可行性。方法：以海藻酸钠-聚赖氨酸-海藻酸钠微胶囊（APA微胶囊）为研究体系，以转内皮抑素重组中国仓鼠卵巢细胞（rCHO）为模型细胞，观察低温保存过程中降温方式、预平衡方式、冷冻保护剂浓度等关键条件对微囊化细胞复苏活性的影响，以及低温保存后微囊膜对IgG的通透性及微囊内细胞的存活、增殖及产物分泌情况。结果：包埋功能细胞的APA微囊经一定时间的低温保存后，微囊膜仍可屏蔽IgG，并未改变其原有的免疫隔离特性，且囊内细胞具有较高的细胞活性，能够正常生存并保持增殖和产物分泌能力。结论：微囊化细胞进行低温保存是可行的。     

APA微囊包裹对牛肾上腺嗜铬细胞分泌儿茶酚胺和亮氨酸脑啡肽的影响

分析海藻酸钠-多聚赖氨酸-海藻酸钠(APA)微囊包裹对牛肾上腺嗜铬细胞(BCCs)分泌儿茶酚胺(CA)和亮氨酸脑啡肽(L-EK)的影响. 用APA微囊包裹原代BCCs, 分别用高效液相色谱-电化学法和放射免疫法检测囊化与非囊化的BCCs分泌CA和L-EK的能力. 与非囊化BCCs相比, 囊化BCCs在单位时间内分泌肾上腺素(E)和去甲肾上腺素(NE)的能力并没有大的改变, 而多巴胺(DA)和L-EK的分泌量则在部分时间点有明显下降. APA微囊对牛肾上腺嗜铬细胞分泌E和NE并无显著的影响, 而对DA和L-EK的分泌有一定影响, 这种影响仅在包裹后短期内发生, 对后期的分泌没有影响.     

微囊化牛肾上腺嗜铬细胞多巴胺含量的检测

目的检测体外培养微囊化牛嗜铬细胞分泌多巴胺的动态变化，为用微囊化肾上腺髓质细胞移植治疗帕金森病提供实验依据。方法常规培养牛肾上腺髓质嗜铬细胞，用人工APA微囊对之微囊化后再培养20d，高效液相色谱(HPLC)动态检测多巴胺含量，并分别在光镜及电镜下观察微囊内牛嗜铬细胞的形态变化。结果牛嗜铬细胞在微囊内生长良好，在培养液内可持续检测到多巴胺。第8d左右多巴胺的分泌达到高峰，随后逐渐下降，14d后分泌量处于一种恒定的状态。结论微囊化牛嗜铬细胞分泌的多巴胺可以通透微囊，可以考虑用来进行异种细胞移植治疗帕金森病。     

APA微囊猪肝细胞异种移植的排斥反应与存活时间研究

目的探讨海藻酸钠-聚赖氨酸-海藻酸钠(APA)微囊猪肝细胞异种移植的排斥反应和存活时间。方法用胶原酶灌注分离猪肝细胞,用海藻酸钠-聚赖氨酸-海藻酸钠法制成微囊肝细胞,将其植入正常SD大鼠腹腔内,两个对照组分别于腹腔植入游离肝细胞和空囊,观察3个月。结果经测定白细胞介素-2、T细胞亚群等免疫指标和组织学检查均无明显变化;经形态学检查显示微囊肝细胞在3个月内有较好活性。结论实验结果表明海藻酸钠-聚赖氨酸-海藻酸钠微囊肝细胞异种移植未发现明显排斥反应且肝细胞存活时间超过3个月。     

APA微囊牛肾上腺嗜铬细胞治疗疼痛的量效时效关系研究

研究海藻酸钠-聚赖氨酸-海藻酸钠(APA)微囊牛肾上腺嗜铬细胞(BCC)治疗大鼠疼痛动物模型的量效时效关系。用结扎坐骨神经制作大鼠疼痛动物模型,将APA微囊BCC移植到动物模型的蛛网膜下腔,分别用冷致痛实验和热过敏实验观察镇痛效果。结果显示与对照组相比,细胞数量5万组、10万组和20万组收缩差异均减小(P<0.05),且随细胞量增加差异减小,表明有明显镇痛作用;40万组与20万组相比没有明显差异。这种镇痛作用可持续12周以上。以上结果表明APA微囊BCC治疗大鼠疼痛动物模型的移植细胞数量和镇痛效果呈正相关关系,有效镇痛时间在12周以上。     

APA-BCC镇痛微囊在癌痛患者脑脊液中的生物学变化

了解海藻酸钙-多聚赖氨酸-海藻酸钙微囊化牛肾上腺嗜铬细胞(APA-BCC微囊)植入癌痛患者脑脊液中的形态、活率及亮氨酸脑啡肽(L-EK)分泌的变化。将APA-BCC微囊按常规腰穿方法植入癌痛患者蛛网膜下腔。7或8天时采取脑脊液,观察APA-BCC的形态、细胞活率,用放射免疫法测定脑脊液中L—EK的含量。移植7天后,患者视觉模拟疼痛评分(VAS)均值由移植前的8．8降为2．4;脑脊液中APA—BCC微囊形态无明显变化;细胞活率由平均91．2％降为89．1％;L-EK含量较移植前增加了1．65倍。将APA-BCC微囊植入癌痛病人脑脊液中能够保持细胞存活、分泌亮啡肽,并产生镇痛效应。     

Percoll非连续等密度梯度离心法纯化牛肾上腺嗜铬细胞

肾上腺髓质嗜铬细胞是具有合成、储存和分泌儿茶酚胺功能的神经元样细胞,对疼痛和帕金森病的治疗具有显著效果。但是,肾上腺髓质嗜铬细胞在体外无增殖能力,只能依赖于肾上腺髓质的分离来获取。牛的肾上腺髓质细胞(Bovine Chromaffin Cells,BCC)由于产量高,易分离获取而成为首选的细胞来源,同时建立一种能够满足临床移植需要的BCC分离纯化方法也显得十分重要。     

微囊化大鼠松果体细胞的体外培养研究

目的 研究微囊化大鼠松果体细胞在体外的生物活性与功能状态。方法 采用胶原酶、胰酶双消化法获取大鼠松果体细胞并进行APA微囊包裹处理，体外培养细胞包囊，相差显微镜观察包囊及细胞形态，台盼蓝染色、5-HT免疫细胞化学法分析包裹细胞活性及分布，高效液相色谱技术(HPLC)检测囊内松果体细胞的褪黑素(MT)分泌状态及微囊的渗透能力。结果 微囊化松果体细胞存活良好，大多数细胞为5-HT免疫反应阳性，HPLC检测证实微囊化松果体细胞MT分泌功能正常，对肾上腺素能激动剂作用敏感。结论 APA细胞微囊具备良好的渗透能力，松果体细胞经微囊包裹处理后其活性和功能未受影响。     

微囊包膜肾上腺组织体外培养研究

为了解微囊包膜大鼠肾上腺组织体外培养存活状况,使用海藻酸钠、氯化钙及多聚-L-赖氨酸制成微囊包膜。取24只大鼠肾上腺组织,其中12只鼠的肾上腺组织用微囊包膜（包膜组）,12只鼠不用包膜（未包膜组）,两组在体外培养36h后,取出培养液;加入促肾上腺皮质激素（ACTH）继续培养24、36h,用放免法分别测ACTH刺激前后的醛固酮、皮质醇浓度。共培养72h取两组肾上腺组织进行光镜和电镜观察。结果提示包膜组醛固酮、皮质醇分泌量显著高于未包膜组（P<0.01）;包膜组经ACTH刺激36h的皮质醇分泌量明显高于刺激前的分泌量（P<0.01）;包膜组经ACTH刺激24、36h醛固酮和皮质醇的分泌量显著高于未包膜组（P<0.01）;未包膜组ACTH刺激前、后的醛固酮、皮质醇分泌量无显著差异（P>0.05）。光镜和电镜观察见肾上腺组织细胞结构完整,细胞存活良好。结论微囊包膜肾上腺组织在体外培养条件下细胞存活良好,微囊不影响组织的分泌功能,并对ACTH刺激有良好的反应。     

Xenotransplantation of microencapsulated bovine chromaffin cells into hemiparkinsonian monkeys

This study examines the effects of xenografts of microencapsulated bovine chromaffin cells (BCCs) on the rotational behavior of hemiparkinsonian monkey recipients. In addition, it determines the content of monoamine neurotransmitters and their major metabolites in the neostriatum in hemiparkinsonian monkeys. The hemiparkinsonian model in monkeys was induced by a unilateral intracarotid injection of methyl-phenyl-tetrahydropyridine (MPTP). Unencapsulated BCCs, BCCs microencapsulated in alginate-polylysine-alginate (ALA) membranes as well as empty microencapsules were grafted into the neostriatum of the hemiparkinsonian monkeys. Following the transplantation the hemiparkinsonian symptoms subsided and the number of rotations induced by apomorphine decreased for up to nine months in the group of recipients grafted with microencapsulated BCCs, while only a temporary improvement (one month) was detected in the recipients of the unencapsulated BCCs. No change was observed in the recipients of empty microencapsules. Dopamine and its metabolites were found considerably depleted in the MPTP-lesioned side versus the unlesioned side of the neostriatum in the hemiparkinsonian monkeys(P<0.05).     

Effect of mouse VEGF164 on the viability of hydroxyethyl methacrylate-methyl methacrylate-microencapsulated cells in vivo: bioluminescence imaging

Bioluminescent imaging was used to track the viability of luciferase transfected L929 cells in poly(hydroxyethyl methacrylate-co-methyl methacrylate) (HEMA-MMA) microcapsules. Bioluminescence, as determined by Xenogen imaging after addition of luciferin to microcapsules in vitro, increased with time, consistent with an increase in cell number. Capsules were suspended in Matrigel and injected subcutaneously. The bioluminesence in vivo increased over the first 3 weeks and then decreased, both with and without the delivery of mVEGF(164) (1.2 ng/24 h/200 microcapsules in vitro); VEGF delivery was from microencapsulated doubly transfected cells (both luciferase and mVEGF(164)). VEGF delivery was sufficient to generate a greater number of vascular structures, but this did not result in the expected increase in microencapsulated cell viability. Interestingly, the number of vessels at day 28 was less than at day 21, consistent with what would be an expected reduction in VEGF secretion when cell viability is lost. The results presented here do not support the hypothesis that transfection of microencapsulated cells with VEGF is sufficient to correct the oxygen transport limitation, at least with this type of tissue engineering construct. On the other hand, bioluminescent imaging proved to be a useful method of monitoring microencapsulated cell viability over many weeks in vivo.     

微囊小肝细胞样前体细胞移植治疗大鼠急性肝衰竭

目的探索大鼠海藻酸钠-聚赖氨酸-海藻酸钠(APA)微囊小肝细胞样前体细胞(SHPC)移植治疗急性肝衰竭(AHF)大鼠模型的可行性和有效性。方法选择体质量150~180 g健康雄性Wistar大鼠10只。采用Retrosine(30 mg/kg)腹腔内注射联合2/3肝切除诱导雄性Wistar大鼠SHPC增殖模型,改进Seglen胶原酶灌注联合Percoll密度梯度离心分离SHPC,倒置显微镜、电子显微镜下观察,并进行免疫组织化学染色;静电液滴法APA微囊包埋。选择体质量180~220 g健康雌性Wistar大鼠20只,随机分为A组(微囊化SHPC腹腔内移植)、B组(空囊对照组);采用D-氨基半乳糖腹腔注射制备大鼠AHF模型;观察模型一般状况及生存时间,术后不同时间点检测血清丙氨酸转氨酶(ALT)、天门冬氨酸氨基转移酶(AST)、总胆红素(TBiL)及血氨水平,检测肝脏病理改变。结果Retrosine腹腔内注射联合2/3肝切除成功建立SHPC增殖模型,分离获得细胞符合大鼠SHPC;APA微囊呈光滑规则球形,直径为(300±40)μm,强度、韧性良好,囊内细胞形态完整,存活率为(90.0±1.3)%。微囊SHPC移植后,A组较B组中位生存时间延长(119.00 h vs 70.33 h),差异有统计学意义(P<0.05);随时间延长,A组大鼠血清ALT、AST、TBiL及血氨水平明显下降,均低于移植前水平,各时间点A组明显低于B组,差异有统计学意义(P<0.05);肝组织病理显示A组肝细胞损伤较B组明显改善;存活48 h大鼠腹腔内微囊保持完整光滑球形,囊内SHPC形态完整,1周微囊有散在破损皱缩,囊内细胞有增多聚集趋势,细胞存活率85%以上。结论APA微囊化大鼠SHPC腹腔内移植能改善大鼠AHF模型生存时间、生物化学指标(ALT、TBiL等)及病理损伤。     

Tumor Anti-angiogenic Gene Therapy with Microencapsulated Recombinant CHO Cells

Microencapsulation of recombinant cells is a novel promising approach to tumor therapy in which therapeutic protein is sustainable and long-term delivered by microencapsulated cells. The semi-permeable membrane of microcapsule can protect cell from host’s immune rejection, increase the chemical stability of therapeutic protein and circumvent the problems of toxicity, limited half-lives and variation in circulating levels. Endostatin, a potent and specific angiogenesis inhibitor, could suppress the growth of primary and metastatic lesions in multiple murine tumor models. In this paper, APA microcapsules with high strength kept intact over 35 days and recombinant CHO cells kept the rapid proliferation viability and the continuous endostatin-expression function. The study of tumor treatment showed that the implantation of microencapsulated recombinant CHO cells decreased the neovascularization of tumor tissue by 59.4% and inhibited the B16 melanoma growth by 77.4%. Twenty days after tumor cell injection, 80% of animals treated with microencapsulated CHO-endo cells were alive compared to only 50% of animals in either control or mock control groups. Therefore, continuous delivery of endostatin from microencapsulated recombinant cells represents a feasible approach to tumor therapy.     

体外培养APA微囊化肿瘤坏死因子α/293细胞对结肠癌细胞增殖的影响

背景：积极探索结肠癌相关基因及抑癌基因已成为新的研究热点,人肿瘤坏死因子α是一个重要的促炎因子和免疫调节因子,对肿瘤的免疫治疗具有一定的疗效。 目的：观察体外培养海藻酸钠-聚赖氨酸-海藻酸钠(alginate-polylysine-alginate,APA)微囊化肿瘤坏死因子α/293细胞对结肠癌细胞增殖的影响。 方法：采用前期建立的制备方法,用APA微囊分别包裹人肿瘤坏死因子α/293细胞,APA微囊化0/293细胞。取对数生长期结肠癌(Lovo)细胞,配制成所需浓度的细胞悬液,接种24孔板培养,分别加入低、中、高剂量稳定转染的APA微囊化肿瘤坏死因子α/293,即分为5个实验组,APA微囊化肿瘤坏死因子α/293细胞低剂量组、中剂量组、高剂量组、阴性组为APA微囊化0/293细胞组,阳性组加入肿瘤坏死因子α,MTT法检测490 nm吸光度;通过对人肿瘤细胞增殖抑制实验,观察对结肠癌细胞(Lovo)增殖的抑制作用。 结果与结论：体外培养中,加入APA微囊化0/293细胞组对结肠癌细胞增殖无抑制作用;而APA微囊化肿瘤坏死因子α/293细胞中、高剂量组和阳性组,在24,48,72 h的A值低于APA微囊化0/293细胞组(P < 0.05),提示APA微囊化人肿瘤坏死因子α/293细胞所分泌肿瘤坏死因子α对结肠癌细胞有增殖抑制效应,均呈现出良好的数量依赖关系。     

APA微囊猪胰岛的实验研究

目的：研究APA微囊猪胰岛的体外体内功能。方法：用胶原酶灌注消化分离猪胰岛，用葡聚糖（dextran）梯度密度离心纯化胰岛，用海藻酸钠．聚赖氨酸．海藻酸钠（APA）包埋胰岛，用静态培养和灌流刺激实验评价猪胰岛体外功能，植入用注射链脲佐菌素（STZ）制作的糖尿病大鼠腹腔评价体内功能。结果：经分离、纯化和包埋后的微囊猪胰岛无论体外静态培养或灌流均能释放胰岛素，并能对培养液中的葡萄糖浓度发生应答。植入糖尿病大鼠腹腔，能降低血糖并分泌胰岛素，维持时间达6个月。结论：APA微囊猪胰岛无论体外体内均能保持较好的功能，对治疗糖尿病动物模型有较好疗效。     

Allotransplantation of Sulphate Glucomannan-Alginate Barium (SGA)-Microencapsulated Rat Islets for the Treatment of Diabetes Mellitus

To offer a more effective microencapsulation technique of islets for the treatment of diabetes, we have developed a new type of microcapsule comprising sulphate glucomannan-alginate barium (SGA). We compared it with traditional microencapsulated APA (alginate-poly-L-lysine-alginate) and ABa (Ba²⁺-alginate) microencapsulated islets. These three types of microencapsulated islets were prepared and cultured in vitro and we studied their morphology and activity. To determine their effects on insulin secretion and cytokine production (MCP-1, IL-1, IFN-γ, TNF-α) the islets were transplanted into diabetic rats. There was no difference in the morphologies of the three types of microencapsulated islets or their insulin secretory capacity in vitro. However, the SGA microencapsulated islets had higher activity and produced more insulin than the APA and ABa microencapsulated islets after transplantation. Normoglycemia was maintained for longer in the SGA-transplanted group than in the other two groups. The concentrations of cytokines in the peritoneal fluid were significantly decreased in the SGA group, as was the infiltration of inflammatory cells around the microcapsules. In conclusion, the novel SGA microencapsulated islets can maintain normoglycemia in diabetic rats without immunosuppression for longer than APA and ABa microencapsulated islets.     

The influence of cellular seeding density in the microencapsulation of hybridoma cells

The aim of this study was to assess the influence of different seeding densities on the function of hybridoma cells (clone 1B5, IgG 2alpha) producing an anti-angiogenic monoclonal antibody (mAb), microencapsulated using a high-voltage electrostatic field. Viable cells were microencapsulated in alginate/poly-L-lysine/alginate (APA) capsules and maintained in tissue culture. Cellular growth rates, production and release of mAb from the capsules were assessed. This study shows that hybridoma cells survive, proliferate and remain functionally competent for over one month in vitro after microencapsulation in APA capsules generated in an electrostatic field. However, the cell seeding density had to be at least 10(7) cells/ml for the microencapsulated cells to be viable and to produce and release mAb through the capsule membrane. The maximum monoclonal antibody concentration in this culture was 29.1 microg/ml by day 17, with a tendency to increase, but capsule breakage impeded the follow-up of this determination.