不同细胞来源松果体微囊功能特性的比较研究

目的：分析体外培养扩增及直接消化分离的松果体细胞微囊的功能差异，筛选最佳的种子细胞来源。方法：游离并摘取乳鼠松果体，胰酶、胶原酶消化分离获取松果体细胞悬液，将直接消化细胞与体外培养一定时间的松果体细胞同时进行海藻酸钠—多聚赖氨酸—海藻酸钠(APA)微囊化处理，采用形态学观察、细胞计数、高效液相色谱(HPLC)技术检测并分析两种细胞微囊的活性和功能差异。结果：体外培养后囊化的松果体细胞分散良好，细胞增殖旺盛，囊内细胞的褪黑素(MT)分泌功能逐渐恢复；直接消化包裹的细胞分散程度较差，有细胞聚集现象，增殖不明显，分泌功能呈下降趋势。结论：体外培养的松果体细胞与微囊材料组织相容性好，囊内细胞活性和功能均优于直接消化包裹的细胞微囊。     

松果体细胞微囊的组织相容性研究

目的：制备松果体细胞微囊并进行动物移植实验，探讨松果体微囊与宿主体的组织相容性。 方法： 游离并摘取乳鼠松果体，胰酶、胶原酶消化分离获取松果体细胞悬液，体外培养后进行海藻酸钠-多聚赖氨酸-海藻酸钠（APA）微囊化处理，将囊化细胞与空囊植入大鼠体内（腹膜腔、肌间隙）。术后15、30 d回收微囊，采用形态学观察、HE染色、细胞计数、高效液相色谱（HPLC）技术检测并分析回收囊的形态、囊内细胞活性、囊壁炎性浸润及纤维化程度，从而判断APA囊膜在体内的组织相容性及其对松果体细胞的免疫保护效应。 结果： 微囊的腹腔回收率约为85％，15 d回收囊大多囊壁完整，少数有破损，囊外粘附巨噬细胞。随植入时间的延长，炎性浸润逐渐加重，囊壁增厚，同一时段微囊与空囊的回收率、囊壁纤维化程度无显著差异。检测证实，15 d回收微囊仍有褪黑素（MT）分泌功能，但细胞功能迅速下降，30 d回收囊的MT检测呈阴性。 结论： APA微囊有一定的组织相容性，对松果体细胞具备免疫保护作用，囊内细胞生存期约为20 d左右，该时段内细胞能维持生物活性和MT分泌功能。     

微囊对大鼠松果体细胞作用的实验研究

目的　探讨微囊化处理后大鼠松果体细胞 5羟色胺 N 乙酰转移酶 (NAT)mRNA的表达及细胞微囊的免疫原性 ,进一步明确人工松果体组织的功能活性与免疫屏蔽效能。　方法　海藻酸钠 聚赖氨酸 海藻酸钠生物微胶囊 (APA微囊 )包裹大鼠松果体细胞 ,采用RT PCR技术检测微囊与对照组松果体细胞的NATmRNA表达 ;运用3H标记胸腺嘧啶核苷 (3H TdR)掺入法比较微囊组、空囊组及游离松果体细胞的淋巴细胞转化值。　结果　不同培养时间对照组和微囊组松果体细胞的NATmRNA表达水平无显著差异 ;微囊组淋巴细胞转化值明显低于游离的松果体细胞 (P <0 0 1) ,但仍高于空囊组 (P <0 0 5 )。　结论　微囊包裹不影响松果体细胞NATmRNA的表达 ,表明APA微囊内松果体细胞代谢正常 ,分泌功能旺盛 ;松果体微囊有一定的免疫保护效应 ,但其隔离作用仍不完全。     

静电法制备小微囊包裹成年猪胰岛的实验研究

目的探索用静电法制备小微囊包裹成年猪胰岛细胞.方法用自制的静电微囊发生装置、制备海藻酸钠-多聚赖氨酸-海藻酸钠(APA)微囊(微囊直径<350μm),包裹成年猪胰岛,体外检测APA小微囊猪胰岛生物活性及微囊膜的通透性.结果静电法制备的APA微囊直径300～350μm,大小相对均一.小微囊包裹成年猪胰岛后,每个微囊内可见1～2个胰岛团,表面光滑.囊内胰岛组织学结构完整,体外培养微囊化胰岛素分泌良好,葡萄糖刺激释放明显,显示了良好的细胞活力及微囊膜通透性.结论用我们自制的静电微囊发生装置能制备APA微囊包裹成年猪胰岛细胞,微囊直径300～350μm,表面光滑,囊内猪胰岛生物活性良好.     

静电法制备小微囊包裹成年猪胰岛的实验研究

目的　探索用静电法制备小微囊包裹成年猪胰岛细胞。方法　用自制的静电微囊发生装置、制备海藻酸钠 -多聚赖氨酸 -海藻酸钠 (APA)微囊 (微囊直径 <35 0 μm) ,包裹成年猪胰岛 ,体外检测APA小微囊猪胰岛生物活性及微囊膜的通透性 .结果　静电法制备的APA微囊直径 30 0～ 35 0 μm ,大小相对均一 .小微囊包裹成年猪胰岛后 ,每个微囊内可见 1～ 2个胰岛团 ,表面光滑 .囊内胰岛组织学结构完整 ,体外培养微囊化胰岛素分泌良好 ,葡萄糖刺激释放明显 ,显示了良好的细胞活力及微囊膜通透性 .结论　用我们自制的静电微囊发生装置能制备APA微囊包裹成年猪胰岛细胞 ,微囊直径 30 0～ 35 0 μm ,表面光滑 ,囊内猪胰岛生物活性良好     

大鼠松果体细胞体外培养及其生物学特性研究

目的 :了解体外培养条件下松果体细胞的形态、生长、增殖及其生物学特性。方法 :无菌技术手术显微镜下剥离、收集乳鼠松果体 ,胰酶消化分离细胞 ,用含 1 0 %小牛血清的DMEM培养液体外培养 ,倒置显微镜观察 ,四唑盐比色测定细胞活性和增殖 ,Hortega染色及 5 -HT免疫细胞化学法鉴定松果体细胞及其功能状态。结果 :体外培养可获得生长旺盛的松果体细胞 ,细胞的生长倍增时间为第 9d ,第 1 0d达高峰。Hortega染色后松果体细胞呈黑色 ,5 -HT免疫阳性细胞占培养细胞的决大部分。在生存期内 ,阳性细胞数量恒定 ,不因培养时间的延长而改变。结论 :体外培养可获得有增殖潜力的松果体细胞 ,细胞在体外功能活跃 ,其活性不因培养时间的延长而降低     

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。     

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。     

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。     

AANAT基因转染成肌细胞及其微囊化研究

目的:构建pTARGETTM-AANAT真核表达载体并转化L6成肌细胞,检测目的基因的表达;制备pTARGETTMMAANAT转基因细胞微囊,初步探讨AANAT转基因细胞微囊的生物活性.方法:RT-PCR技术扩增大鼠AANAT cDNA,酶切连接pTARGETTM载体,脂质体法转染L6细胞并检测pTARGETTM-AANAT的基因表达产物;将高表达活性的L6细胞进行APA微囊化处理,倒置显微镜观察微囊形态,Western-blot检测囊内细胞AANAT蛋白表达活性.结果:酶切、DNA测序及凝胶电泳检测结果证实,pTARGETTM-AANAT真核表达载体构建成功.转染后的L6细胞表达AANAT mRNA,胞浆中能检测到从NAT蛋白的表达.转染细胞微囊体外存活良好,微囊外形完整无粘连,大小较均一,囊内细胞清晰可见,体外培养两周后破囊检测证实囊内细胞具有AANAT蛋白表达活性.结论:pTARGETTM-AANAT真核表达载体在L6细胞中成功表达,转染细胞微囊体外存活良好,囊内细胞具备AANAT蛋白表达活性.     

Microencapsulation and culture in vitro of rat pinealocytes

BACKGROUND: Melatonin is a powerful anti-aging reagent for scavenging free radicals. However, the effect of exogenous melatonin on age-dependent diseases is uncertain. Immune rejection has limited xenotransplantation or allotransplantation of the pineal gland. The aim of this study was to assess cell viability and the function of rat pinealocytes encapsulated in APA capsules and offer experimental suggestions for pineal microencapsulation grafting to resist aging. METHODS: The pineal glands of neonatal rats were removed. Pinealocytes were isolated and encapsulated in APA microencapsulation and cultured. Morphological appearance of the microencapsulation was observed. Trypan blue staining and 5-HT immunocytochemical assay were used to detect cell viability and identify pinealocytes. The expression of AA-NAT mRNA was confirmed by RT-PCR. Melatonin release was measured and compared by HPLC. RESULTS: Both control and encapsulated pinealocyte cultures survived well. The majority of the encapsulated pinealocytes as well as unencapsulated cells remained 5-HT positive. No significant difference in melatonin secretion and the expression level of AA-NAT mRNA between encapsulated and unencapsulated pinealocytes was found. CONCLUSIONS: Pinealocytes survive and remain functionally competent in vitro at least 2 weeks after microencapsulation.     

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.     

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 2 α) 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 10⁷ 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 μg/ml by day 17, with a tendency to increase, but capsule breakage impeded the follow-up of this determination.     

Xenotransplantation of exendin-4 gene transduced pancreatic islets using multi-component (alginate,poly-L-lysine,and polyethylene glycol) microcapsules for the treatment of type 1 diabetes mellitus

This study proposed that microencapsulation of exendin-4 gene transduced islets using alginate, poly-L-lysine, and polyethylene glycol could lead to increased viability and functionality of islets in a rat to mouse xenograft model. The stability of the microcapsules was determined using an osmotic pressure test and a rotational stress test. Exendin-4 gene was transduced into pancreatic islets using lenti-viral vectors and the transduced islets were encapsulated using multi-component microcapsules mentioned above. Both viability and functionality of microencapsulated islets were evaluated in both in vitro and in vivo xenograft model. The viabilities of the unmodified islets (control) and the exendin-4 transduced islets (test) on 14th day were 18.6?±?11.1 and 49.2?±?13.4%, respectively (p?<?0.05). The stimulation index of the control and the test groups was 2.3?±?1.7 and 3.0?±?1.6, respectively. The mean survival times (MST) of the control and the test groups were 20.2?±?8.0 and 35.2?±?10.0?days, respectively (p?<?0.05). Significant differences in MST suggested that transduction of exendin-4 gene had a great potential to increase the function of encapsulated islets. In conclusion, exendin-4 gene transduced islets encapsulated by poly(ethylene glycol) conjugated alginate/PLL microcapsules significantly improved both viability and functionality of encapsulated islets.     

Poly(methyl vinyl ether-alt-maleic acid) polymers for cell encapsulation

Polyanions based on poly(methyl vinyl ether-alt-maleic acid) were investigated as materials for cell encapsulation. These water-soluble polyanions having molecular masses ranging from 20 to 1980 kDa were prepared by functionalization of poly(methyl vinyl ether-alt-maleic anhydride) with 5-aminofluorescein and/or α-methoxy-ω-amino-poly(ethylene glycol), followed by base hydrolysis of the residual anhydride groups to form the corresponding poly(methyl vinyl ether-alt-sodium maleate). Their potential to replace alginate both in the core and, in particular, the outer shell of calcium alginate-poly(L-lysine)-alginate (APA) capsules was determined using confocal fluorescence microscopy, osmotic pressure tests, permeability studies, protein binding and cell viability assays. These polymers were shown to be able to replace the outer layer of alginate, forming more resilient capsule shells. The resulting capsules showed similar permeability and resistance to bovine serum albumin binding, as well as superior viability for encapsulated cells, when compared to standard APA capsules. In addition, these polymers showed promise for use as functional additives to the capsule cores.     

永生化下颌骨髁突软骨细胞的微囊化研究

为了探讨微囊包裹软骨细胞在软骨组织工程中的适用性 ,根据气流切割原理采用海藻酸钠 -多聚赖氨酸 -海藻酸钠 ( APA)对永生化下颌骨髁突软骨细胞 ( Im mortalized mandibular condylar chondrocyte,IMCC)进行微囊包裹。用倒置显微镜观察、台盼蓝染色、细胞记数、HE染色、免疫组化等方法检测微囊的大小、细胞的生长及微囊内组织的软骨特性等情况。研究发现 ,IMCC可在微囊内存活 ,活细胞率 >80 % ,微囊直径平均 779μm。细胞数量随着培养时间的延长逐渐增多 ,约 2 0 d左右达到平台期 ,细胞在囊内呈簇样生长 ,高表达软骨特异的蛋白多糖和 型胶原。提示 IMCC可在微囊内形成类软骨组织样结构 ,微囊技术适用于包裹软骨细胞     

Alginate-based microencapsulation of retinal pigment epithelial cell line for cell therapy

The goals of this study were to evaluate human retinal pigment epithelial cell line (ARPE-19) for cell encapsulation and to optimize the alginate-based microencapsulation. We used immortalized ARPE-19 cells and the transfected sub-line that expresses secreted alkaline phosphatase (SEAP) reporter enzyme. Alginate was cross-linked with different divalent cations (Ca(2+), Ba(2+), Sr(2+) and combination of Ca(2+) and Ba(2+)), coated first with poly-l-lysine (PLL), and then with alginate. Microcapsules with different pore sizes and stability were generated. The pore size of the microcapsules was assessed by the release of encapsulated fluorescein isothiocyanate (FITC)-dextrans. The viability of the cells in the microcapsules was studied in vitro by assessing the secretion rates of SEAP and oxygen consumption by the cells. The best microcapsule morphology, durability and cellular viability were obtained with alginate microcapsules that were cross-linked with Ca(2+) and Ba(2+) ions and then coated with PLL and alginate. Based on FITC-dextran release these microcapsules have porous wall that enables the rapid contents release. The ARPE-19 cells maintained viability in the Ca(2+) and Ba(2+) cross-linked microcapsules for at least 110 days. The alginate microcapsules cross-linked with Ca(2+) and Ba(2+) have sufficiently large pore size for prolonged cell viability and for the release of secreted SEAP model protein (Mw 50 kDa; radius of gyration of 3 nm). ARPE-19 cells show long-term viability and protein secretion within alginate microcapsules cross-linked with Ca(2+) and Ba(2+). This combination may be useful in cell therapy.     

X-ray-visible microcapsules containing mesenchymal stem cells improve hind limb perfusion in a rabbit model of peripheral arterial disease

The therapeutic goal in peripheral arterial disease (PAD) patients is to restore blood flow to ischemic tissue. Stem cell transplantation offers a new avenue to enhance arteriogenesis and angiogenesis. Two major problems with cell therapies are poor cell survival and the lack of visualization of cell delivery and distribution. To address these therapeutic barriers, allogeneic bone marrow-derived mesenchymal stem cells (MSCs) were encapsulated in alginate impregnated with a radiopaque contrast agent (MSC-Xcaps). In vitro MSC-Xcap viability by a fluorometric assay was high (96.9% ± 2.7% at 30 days postencapsulation) and as few as 10 Xcaps were visible on clinical x-ray fluoroscopic systems. Using an endovascular PAD model, rabbits (n = 21) were randomized to receive MSC-Xcaps (n = 6), empty Xcaps (n = 5), unencapsulated MSCs (n = 5), or sham intramuscular injections (n = 5) in the ischemic thigh 24 hours postocclusion. Immediately after MSC transplantation and 14 days later, digital radiographs acquired on a clinical angiographic system demonstrated persistent visualization of the Xcap injection sites with retained contrast-to-noise. Using a modified TIMI frame count, quantitative angiography demonstrated a 65% improvement in hind limb perfusion or arteriogenesis in MSC-Xcap-treated animals versus empty Xcaps. Post-mortem immunohistopathology of vessel density by anti-CD31 staining demonstrated an 87% enhancement in angiogenesis in Xcap-MSC-treated animals versus empty Xcaps. MSC-Xcaps represent the first x-ray-visible cellular therapeutic with enhanced efficacy for PAD treatment.