Construction and identification of immortalized rat astrocyte cell line expressing enkephalin

Objective ： To provide a sound cell source for further ex-vivo gene therapy for chronic pain, we attempt to develop an immortalized rat astrocyte cell line that expresses enkephalin regulated by doxycycline. Methods. Retrovirus infection method was employed to develop an immortalized rat astrocyte cell line that could express enkephalin regulated by doxycycline. The hPPE gene expression level of immoralized astroyte cells （IAC）/ hPPE was detected by RT-PCR, indirect immunofluorescence staining and radioimmunoassay. Results. IAC carrying Tet-on system transfected with preproenkephalin gene could secrete enkephalin that was regulated by doxycycline in a dose-dependent manner and hPPE gene activation could be repeated in on-off-on cycles through administration or removal of doxycycline. Conclusion： An immortalized rat astrocyte cell line that secrete enkephalin under the control of doxycycline is established successfully, which provides a research basis for transgenic cell transplantation for analgesia.     

Intrathecal grafting of porcine chromaffin cells reduces formalin-evoked c-Fos expression in the rat spinal cord

Chromaffin cells from the adrenal gland secrete a combination of neuroactive compounds including catecholamines, opioid peptides, and growth factors that have strong analgesic effects, especially when administered intrathecally. Preclinical studies of intrathecal implantation with xenogeneic bovine chromaffin cells in rats have provided conflicting data with regard to analgesic effects, and recent concern over risk of prion transmission has precluded their use in human clinical trials. We previously developed a new, safer source of adult adrenal chromaffin cells of porcine origin and demonstrated an in vivo antinociceptive effect in the formalin test, a rodent model of tonic pain. The goal of the present study was to confirm porcine chromaffin cell analgesic effects at the molecular level by evaluating neural activity as reflected by spinal cord c-Fos protein expression. To this end, the expression of c-Fos in response to intraplantar formalin injection was evaluated in animals following intrathecal grafting of 10(6) porcine or bovine chromaffin cells. For the two species, adrenal chromaffin cells significantly reduced the tonic phases of the formalin response. Similarly, c-Fos-like immunoreactive neurons were markedly reduced in the dorsal horns of animals that had received injections of xenogeneic chromaffin cells. This reduction was observed in both the superficial (I-II) and deep (V-VI) lamina of the dorsal horn. The present study demonstrates that both xenogeneic porcine and bovine chromaffin cells transplanted into the spinal subarachnoid space of the rat can suppress formalin-evoked c-Fos expression equally, in parallel with suppression of nociceptive behaviors in the tonic phase of the test. These findings confirm previous reports that adrenal chromaffin cells may produce antinociception by inhibiting activation of nociceptive neurons in the spinal dorsal horn. Taken together these results support the concept that porcine chromaffin cells may offer an alternative xenogeneic cell source for transplants delivering pain-reducing neuroactive substances.     

强力霉素调控表达脑啡肽的永生化大鼠星形胶质细胞株的构建

目的构建受强力霉素（Dox）调控表达脑啡肽的永生化大鼠星形胶质细胞株（IAST）。方法采用脂质体介导法将重组质粒pRevTREhPPE和调节质粒pRevTet-On分别转染逆转录病毒包装细胞PT67；将含有RevTet—On和RevTRE／hPPE病毒上清感染IAST，得到稳定表达脑啡肽的IAST／Tet-On／hPPE细胞株，实时定量PCR检测Dox定量调控该细胞株前脑啡肽原（hPPE）基因的表达，免疫细胞化学及放射免疫分析法检测Dox定量调控该细胞株中脑啡肽的表达。结果IAST／Tet-On／hPPE细胞株中，hPPE基因的表达和脑啡肽的分泌受Dox调控，Dox浓度为100～5000nedml时，随着Dox浓度增高，hPPE基因的表达和脑啡肽的分泌增加，Dox浓度为5000ng／ml时达峰值。结论成功构建了受四环素及其衍生物强力霉素定量调控表达脑啡肽的IAST。     

One-Year Chromaffin Cell Allograft Survival in Cancer Patients With Chronic Pain: Morphological and Functional Evidence

The control of chronic pain through transplantation of chromaffin cells has been reported over the past few years. Analgesic effects are principally due to the production of opioid peptides and catecholamines by chromaffin cells. Clinical trials have been reported with allografts consisting of whole-tissue fragments implanted into the subarachnoid space of the lumbar spinal cord 14, 19, 36. In the present study, allogeneic grafts were successfully used to control chronic pain in two patients over a period of 1 yr based on patient reported pain scores, morphine intake, and CSF levels of Met-enkephalin. Macroscopic examination at autopsy located the transplanted tissue fragments in the form of multilobulated nodules at the level of the spinal axis and cauda equina. Immunocytochemical microscopy showed neuroendocrine cells are positive for chromagranin A (CGA), and enzymes tyrosine hydroxylase (TH) and dopamine-β-hydroxylase (DβH). The results suggest that there is a relationship between analgesic effect, Met-enkephalin levels in CSF, and the presence of chromaffin cells surviving in spinal subarachnoid space.     

人前脑啡肽原基因在体细胞系细胞的表达

目的 检测人前脑啡肽原基因（ｈｐｐｅ）鼠、犬,人体细胞系细胞的表达水平,评估转脑啡肽基因细胞系用于细胞镇痛的可行性。方法 采用磷酸钙共沉淀法或脂质体包裹法,将重组质粒ｐＣＭＶｈＰＰＥ（ｐＥ）单独或与ｐＲＳＶｎｅｏ（ｐＮ）共转染导入三种哺乳动物体细胞系细胞（ＮＩＨ／３Ｔ３,ＭＤＣＫ,ＷＩＳＨ）中,用Ｇ４１８培养液筛选阳性共转染细胞,扩增培养。以放射免疫法测定细胞培养上清液中脑啡肽（ＥＮＫ）的含量。结     

Intrathecal implants of microencapsulated xenogenic chromaffin cells provide a long-term source of analgesic substances

Adrenal medullary chromaffin cells secrete several neuroactive substances including catecholamines and opioid peptides that produce analgesic effects in the central nervous system. This study was designed to investigate whether intrathecal microencapsulated chromaffin cells could release analgesic materials producing antiallodynic effects on the chronic neuropathic pain in rats induced by chronic constriction injury (CCI) of the sciatic nerve. Prior to intrathecal implantation, chromaffin cells were encapsulated with alginate and poly-L-lysine to protect them from the host immune system. Behavior tests were performed before CCI, 1 week later, and at 4, 7, 14, 21, 28 days postimplantation. At the end of study, we performed cerebrospinal fluid (CSF) collection and implant retrieval. We observed that intrathecal implantation of encapsulated xenogenic chromaffin cells reduced the mechanical and cold allodynia in a model of neuropathic pain. CSF levels of catecholamines and metenkephalin in the rats that received implants were higher than the controls. In addition, we observed chronic survival of implants. These results suggested that intrathecal microencapsulated chromaffin cells may represent a new approach to chronic neuropathic pain management.     

人胎儿表皮干细胞的体外分离培养及基因转染

目的：探讨人胎儿表皮干细胞体外分离培养的方法以及作为体外基因转染靶细胞的可行性。方法：利用Ⅳ型胶原快速贴附法分离人胎儿表皮干细胞，以人胎儿成纤维细胞条件培养液配制表皮干细胞培养基，通过角蛋白19（K19）和整合素β1免疫组化染色、细胞周期分析及克隆形成率测定，对培养细胞进行鉴定。采用脂质体介导法，以含血管内皮细胞生长因子165（VEFG165）基因片段的真核表达载体pcDNA3.1(pcDNA3.1/VEGF165)转染培养细胞；采用病毒载体介导法，以含报告基因绿色荧光蛋白（GFP）的重组腺相关病毒载体（raav/GFP)转染培养细胞。应用免疫组化染色及荧光显微镜观察检测转染效果。结果：人胎儿表皮干细胞呈明显克隆性生长、克隆形成率高，G1期细胞比例明显高于普通基底层角质细胞，K19和整合素β1免疫组化染色呈强阳性。pcDNA3.1/VEGF165转染的表皮干细胞VEGF165免疫组化染色阳性，raav/GFP转染的表皮干细胞呈现强荧光。结论：利用Ⅳ型胶原快速贴附法及人胎儿成纤维细胞条件培养基，可初步实现人胎儿表皮干细胞的分离培养。以质体为介导或以腺相关病毒为载体进行人胎儿表皮干细胞的体外基因转染是可行的。     

Lipofectamine介导转染神经干细胞的研究

目的观察阳离子脂质体法转染体外培养神经干细胞的转染效率和外源基因的表达。方法从1d龄新生鼠大脑皮层组织培养神经干细胞，带有报告基因GFP的穿梭质粒pAd．Tract-CMV经Lipofectamine介导转染神经干细胞后观察GFP表达，用流式细胞仪测定转染率。并观察阳离子脂质体对神经干细胞的毒性作用。结果荧光显微镜观察到被转染的神经干细胞长期表达绿色荧光蛋白。流式细胞仪结果显示转染率最高可达到39．99％。转染时，阳离子脂质体浓度超过24ml／L时表现出细胞毒性。结论阳离子脂质体hpofectamine介导转染神经干细胞效率较高，外源基因表达时间长。     

Viability and functionality of bovine chromaffin cells encapsulated into alginate-PLL microcapsules with a liquefied inner core

Implantation of adrenal medullary bovine chromaffin cells (BCC), which synthesize and secrete a combination of pain-reducing neuroactive compounds including catecholamines and opioid peptides, has been proposed for the treatment of intractable cancer pain. Macro- or microencapsulation of such cells within semipermeable membranes is expected to protect the transplant from the host's immune system. In the present study, we report the viability and functionality of BCC encapsulated into microcapsules of alginate-poly-L-lysine (PLL) with a liquefied inner core. The experiment was carried out during 44 days. Empty microcapsules were characterized in terms of morphology, permeability, and mechanical resistance. At the same time, the viability and functionality of both encapsulated and nonencapsulated BCC were evaluated in vitro. We obtained viable BCC with excellent functionality: immunocytochemical analysis revealed robust survival of chromaffin cells 30 days after isolation and microencapsulation. HPLC assay showed that encapsulated BCC released catecholamines basally during the time course study. Taken together, these results demonstrate that viable BCC can be successfully encapsulated into alginate-PLL microcapsules with a liquefied inner core.     

人前脑啡肽原基因修饰人骨髓间充质干细胞系的构建

目的 构建人前脑啡肽原(PENK)基因修饰的并可稳定分泌脑啡肽蛋白的人骨髓间充质干细胞系(hMSCs).方法 采用脂质体法将PENK基因逆转录病毒载体质粒(pBABE-PENK)转染至Phoenix-293T细胞,收集病毒上清液感染hMSCs细胞,经过嘌呤霉素筛选得到稳定表达PENK基因的hMSCs细胞株(hMSC-PENK细胞).以转染空载体细胞作为对照,即hMSC-pBABE细胞.采用RT-PCR法检测PENK mRNA的表达,免疫荧光法测定亮氨酸脑啡肽(LEK)的表达,ELISA法测定细胞培养上清液LEK浓度.结果 与hMSCs细胞和hMSC-pBABE细胞比较,hMSC-PENK细胞PENK mRNA和LEK表达上调,细胞培养上清液中LEK浓度升高(P＜0.05或0.01).结论 PENK基因修饰的hMSCs可表达PENK基因并分泌脑啡肽蛋白,成功构建了稳定分泌镇痛物质的细胞系.     

Reduction of Allodynia by Intrathecal Transplantation of Microencapsulated Porcine Chromaffin Cells

Bovine chromaffin cells (BCCs) are well known to have analgesic effect to reduce acute or chronic pain when transplanted in the subarachnoid space and have been considered as an alternative therapy for pain management. However, due to recent concerns over risks associated with prion transmission, porcine tissue is considered to be an alternate xenogeneic source for clinical use. In the present study, we investigated whether microencapsulated porcine adrenal medullary chromaffin cells (PCCs) also have analgesic effect to reduce allodynia caused by neuropathic pain in chronic constriction injury model of rat. PCCs were isolated from a porcine adrenal medulla and then microencapsulated with alginate and poly. In in vitro tests, the microencapsulated PCCs were investigated whether they have an ability to release catecholamines responding to nicotine stimulation. The levels of catecholamines released from the microencapsulated PCCs were significantly higher than from microencapsulated BCCs. In addition, the microencapsulated PCCs released catecholamines and met-enkephalin responding to cerebral spinal fluid (CSF) retrieved from a neuropathic pain model. In in vivo tests, implantation of microencapsulated PCCs reduced both mechanical and cold allodynia in chronic constriction injury model of a rat whereas the microencapsulated BCCs reduced only cold allodynia under the same conditions. The injection of antagonist of opioid peptides reversed the reduction of cold allodynia in microencapsulated PCC-received animal. The levels of catecholamines in the CSF of rats after implantation of microencapsulated PCCs were significantly higher than in the control group. These data suggest that microencapsulated PCCs may be another effective source for the treatment of neuropathic pain.       

人β干扰素基因重组腺病毒载体的构建及在骨髓间充质干细胞的表达

目的 构建人β干扰素(hIFN-β)基因重组腺病毒,观察重组腺病毒介导的hIFN-β转染大鼠骨髓间充质干细胞(MSCs)后,细胞内hIFN-β表达情况以及重组腺病毒对MSCs生长的影响.方法 利用细菌内同源重组技术快速构建Ad-hIFN-β腺病毒重组质粒,经酶切及测序鉴定正确后转染人胚肾细胞HEK293包装成为重组Ad-hIFN-β腺病毒,并进行滴度测定.转染的MSCs行逆转录-聚合酶链反应(RT-PCR)检测细胞内hIFN-β mRNA的表达;酶联免疫吸附试验(ELISA)法检测培养液上清hIFN-β的分泌情况;噻唑蓝(MTF)比色法检测细胞活力并绘制转染后MSCs生长曲线.结果 经限制性内切酶检测、基因测序及和绿色荧光观察证实成功的构建了携带hIFN-β基因的重组腺病毒,且重组腺病毒滴度高达1×109pfu/ml.Ad-hIFN-β转染MSCs后在荧光显微镜下证实有绿色荧光;RT-PCR证明转染的MSCs内有hIFN-β mRNA的表达;ELISA检测转染组1、3、5、7、10、15、20 d上清液中hIFN-β蛋白分泌量分别为192、273.436、957、605、472、279 ng/L,明显高于对照组(P<0.01);Ad-hIFN-β转染的MSCs生长曲线与正常培养MSCs差异无统计学意义(P>0.05).结论 成功构建了携带hIFN-β基因的重组腺病毒载体,重组腺病毒转染对MSCs的增殖能力无明显影响,而且Ad-hIFN-β转染大鼠MSCs能够表达并分泌hIFN-β蛋白.     

Automated method for isolation of adrenal medullary chromaffin cells from neonatal porcine glands

An automated method for the isolation of neonatal porcine adrenal chromaffin cells is described. Adrenal chromaffin cells are potentially useful for therapeutic transplantation, but current isolation methodology suffers from labor intensiveness and variability in yield and viability due to imprecision of manual techniques, enzyme purity, and gland age and species. The described approach utilizes an adaptation of an automated procedure previously described for isolation of pancreatic islets. Results from neonatal porcine adrenal glands revealed consistent cell yields with high (approximately 99%) viability. Catecholamine assays showed that the resultant cultures continue to produce and secrete norepinephrine and epinephrine. Immunocytochemical analysis indicated that the majority of cells in the preparation are chromaffin cells and adrenal cortical cells. The procedure meets the following requirements: 1) minimal traumatic action on the adrenal chromaffin cells, 2) continuous digestion in which the adrenal cells that are progressively liberated can be saved from further mechanical action, 3) minimal human intervention in the digestion process, and 4) high yield and viability of the isolated adrenal chromaffin cells.     

绿色荧光蛋白体外转染与体内示踪成骨细胞的研究

目的观察经腺病毒介导的绿色荧光蛋白(greenfluorescentprotein,GFP)转染的成骨细胞的体外表达及体内示踪情况,探讨GFP作为组织工程种子细胞示踪剂的可行性。方法以腺病毒为载体,293A细胞为包装细胞,介导GFP转染成人骨髓源成骨细胞,与未转染的同期细胞作对照,在相差显微镜和荧光显微镜下观察,流式细胞术检测GFP表达效率;分别检测转染后两组细胞的碱性磷酸酶(ALP)活性与骨钙素(OCN)的含量;并将GFP转染8d后的成骨细胞植入裸鼠股部肌袋内,术后4、8周取材进行荧光显微镜、HE染色组织学和免疫组织化学染色观察。结果GFP转染的骨髓源成骨细胞表达绿色荧光的阳性率达75%以上,转染8d后的成骨细胞表面抗原标志CD29、CD44高效表达,而CD34不表达;转染后4、8d细胞内ALP活性与OCN含量与未转染组比较差异无显著性意义(P>005)。GFP转染8d后的成骨细胞植入裸鼠体内4、8周均可表达明显的绿色荧光,并具有成骨细胞的形态特征,ALP免疫组化染色呈黄褐色。结论GFP能在体外转染、裸鼠体内示踪成骨细胞,是一种可用于组织工程研究的理想的活细胞示踪剂。     

慢病毒载体介导绿色荧光蛋白基因在椎间盘髓核细胞中的表达

目的本研究构建含绿色荧光蛋白基因的重组慢病毒,并观察其在人椎间盘髓核细胞中的表达情况。方法应用分子克隆技术将绿色荧光蛋白（green fluorescent protein,GFP）基因导人慢病毒载体质粒pLenti6／V5 TOPO,应用磷酸钙沉淀法将慢病毒载体三质粒系统（包括包装质粒、包膜蛋白质粒等）共转染入293细胞进行包装,48h后在荧光显微镜下观察绿色荧光蛋白表达情况,72h收集病毒上清并感染髓核细胞,在荧光显微镜下感染情况。结果重组慢病毒滴度测定约为10^7U／mL。感染人椎间盘髓核细胞后,荧光显微镜下观察到GFP的表达。结论成功构建了含GFP的慢病毒载体,且能成功将目的基因转入椎间盘髓核细胞并表达。     

异体嗜铬细胞蛛网膜下腔移植治疗晚期癌痛病人的可行性

目的 评价异体嗜铬细胞蛛网膜下腔移植治疗晚期癌痛的镇痛效果和安全性,方法 选择经常规方法治疗仍不能缓解的晚期癌痛病人１０例,随机分为两组,试验组（ｎ＝４）经蛛蛛网膜下腔注入体外培养３ｄ的细胞县液２ｍｌ;对照组（ｎ＝６）注入同体积细胞培养液,移植后继续应用阿片制剂。移植前、后观察病人的疼痛程度（ＶＡＳ法）和阿片制剂日摄量,分别采用高效液相色谱法及放射免疫技术测定脑脊液儿茶酚胺和脑啡肽的浓度以及采用免疫技术测定ＡＴ淋巴细胞亚群及血清抗体和补体水平。结果 与移植前比较,两组病人的ＶＡＳ值均显著性降低（Ｐ〈０．０５或０．０１）;试验组阿片制剂日摄量移植后有显著性减少（Ｐ〈０．０５或０．０１）,对照组无明显变化;两组病人脑脊液儿茶酚胺浓度均显著性升高（Ｐ〈０．０１）,脑啡肽浓度均显著性降低（Ｐ〈０．０１）;两组病人的血清     

肿瘤靶向DNA载体转铁蛋白-多聚乙烯亚胺体外转染

目的评价转铁蛋白一多聚乙烯亚胺（Tf—PEI）靶向性转染小鼠骨肉瘤细胞的可行性。以Tf—PEI为载体，将小鼠白细胞介素（IL）-12基因导人小鼠骨肉瘤细胞。方法以Tf-PEI为载体，将绿色荧光蛋白基因和小鼠IL-12基因分别导人小鼠骨肉瘤细胞，并检测其转染效率。以游离转铁蛋白竞争性拮抗Tf-PEI，并观察它对转染效率的影响。结果Tf-PEI可以高效的、靶向性的转染小鼠骨肉瘤细胞。当N／P比值为5时，Tf-PEI的转染效率最高。游离转铁蛋白能够显著的拮抗Tf-PEI的转染。Tf-PEI还可以成功的将小鼠IL-12基因导人小鼠骨肉瘤细胞。结论 Tf—PEI是一种高效率、低毒性、肿瘤靶向性的基因转染载体，可以广泛应用于恶性肿瘤靶向性基因治疗的试验。     

Immunolocalization of cystinosin,the protein defective in cystinosis

Cystinosis is an autosomal recessive disorder associated with excessive lysosomal cystine accumulation secondary to defective lysosomal cystine efflux. CTNS, the gene mutated in cystinosis, codes for the lysosomal membrane protein cystinosin. Antisera were raised in rabbits to a carboxy-terminal oligopeptide sequence from cystinosin. Antisera were screened by Western blotting and immunocytochemical analyses of transfected COS-7 cells expressing either human wild-type cystinosin, a wild-type cystinosin-green fluorescent protein (GFP) fusion protein, or a fusion protein of GFP and mutant human cystinosin with a carboxy-terminal deletion. In Western blots, bands corresponding to cystinosin or cystinosin-GFP were observed in transfected cells but no signal was detected in cells expressing the carboxy-terminal mutant; preimmune sera yielded negative results in all three cases. In transfected cells expressing wild-type cystinosin, immunoreactivity appeared in subcellular vesicles. In cells expressing the wild-type cystinosin-GFP fusion protein, immunoreactivity colocalized with GFP fluorescence. Previous studies demonstrated that GFP fluorescence from this construct colocalized with immunostaining for a known lysosomal membrane protein, i.e., lysosome-associated membrane protein 2. In immunohistochemical analyses, cystinosin localized to tubule epithelia in three normal human kidneys, with a pattern similar to that of lysosome-associated membrane protein 2; cystinosin immunoreactivity was absent in kidneys from patients with a CTNS deletion. For the first time, antisera have been raised that localize cystinosin in cells in vitro and in vivo.     

Ad-BMP-2/GFP转染兔骨髓间充质干细胞前后生物学特性的研究

目的：对Ad－BMP－2／GFP转染兔骨髓间充质干细胞（ BMSCs ）前后的生物学特性进行观察。方法采用密度梯度离心结合贴壁培养法分离获取第10代兔BMSCs,应用流式细胞仪检测细胞表面抗原CD44、CD45、CD29的表达,用携带BMP2和GFP基因的腺病毒转染细胞。分别通过倒置荧光显微镜下观察转染前后细胞形态改变,MTT 法分析转染前后细胞增殖的情况,体外Ⅰ型胶原免疫组化染色和钙结节茜素红染色以观察转染前后细胞的成骨分化情况,Western Blot 检测转染前后细胞内目的蛋白表达。结果密度梯度离心结合贴壁培养法能获取高纯度第10代兔BMSCs ,流式细胞仪检测显示CD44、CD29阳性,CD45阴性。 Ad－BMP－2／GFP转染兔BMSCs后在荧光显微镜下观察到绿色荧光,细胞形态向成骨方向分化,在短时间内能促进兔BMSCs 的增殖（P＜0．05）,Ⅰ型胶原免疫组化染色、茜素红染色均呈阳性,Western Blot显示细胞内稳定表达BMP－2目的蛋白。结论 Ad－BMP－2／GFP能成功转染兔骨髓间充质干细胞并能改变其生物学特性。     

人胚肾细胞SGLT2基因异源表达体系的构建

目的 构建钠依赖的葡萄糖转运蛋白2（SGLT2）基因异源表达体系,为探讨突变引起SGLT2蛋白功能及表达异常的分子机制提供实验依据。 方法 利用RT-PCR法从人肾组织中获得SGLT2基因,将该基因克隆到真核表达载体PEXL-GFP（绿色荧光蛋白）中,将携带有SGLT2基因的PEXL载体转染人胚肾细胞系（HEK293细胞）,获得目的蛋白的瞬时表达。应用Western印迹及激光共聚焦显微镜检测融合蛋白在HEK293细胞的表达和分布,并通过摄取实验进一步验证SGLT2蛋白的转运功能。 结果 SGLT2-GFP蛋白可在HEK293细胞表达,激光共聚焦显微镜观察发现,SGLT2-GFP蛋白在细胞膜上呈点状分布,并且与细胞膜标记物（DiI）有良好的共定位,转染SGLT2-GFP质粒的HEK293细胞的转运活性较对照组（未转染及转染空质粒细胞组）强约3.5倍（P < 0.01）。 结论 成功构建了SGLT2真核表达载体,为探讨SGLT2基因表达、功能及SGLT2突变在家族性肾性糖尿发病的遗传机制提供了重要的依据。