小鼠类胚胎干细胞来源的转基因前体细胞构建动物乳腺生物反应器的研究
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摘要
自胚胎干细胞(Embryonic stem cells,ES细胞)问世以来,ES细胞技术就一直被认为是建立动物乳腺生物反应器的最佳方法。但是奶用家畜的(类)ES细胞系不易建立,其培养技术难以达到建立乳腺生物反应器的要求。此外,现行的乳腺生物反应器的建立往往是通过转基因动物途径实现的,该生产模式难度高、效率低、时间长而且费用高,限制其推广普及。因此,探求一种基于ES细胞的快速建立动物乳腺生物反应器的新方法显得尤为重要。
体外定向分化的研究一直是干细胞领域研究的热点,理论上ES细胞可以分化为200多种类型的细胞,目前已经获得了ES细胞来源的神经前体细胞、胰岛细胞、角质细胞、上皮前体细胞、心肌细胞、生殖细胞等多种类型的细胞。研究发现,将这些ES细胞来源的前体/干细胞移植后可以在宿主体内微环境诱导下进一步分化为特定细胞,并与周围组织整合,发挥正常生理功能。经过基因修饰后的乳腺上皮细胞被移植到宿主乳腺组织后可长期生存并表达外源基因。因此,利用ES细胞来源的前体/干细胞移植策略结合基因修饰技术构建动物乳腺生物反应器是可行的:以小鼠为动物模型,将稳定转染目的基因(由山羊乳球蛋白基因(β-lactoglobulin,BLG)调控的)的小鼠类ES细胞来源的上皮前体细胞移植到同源小鼠的乳腺组织,随着移植细胞在宿主乳腺组织生存、整合并发挥正常的生理功能,受体动物的乳腺可以分泌含有目的蛋白的乳汁,最终达到快速建立乳腺生物反应器模型的目的。
本研究将昆明白小鼠早期类ES细胞在体外定向分化为上皮前体细胞,通过稳定转染的方式,将携带编码猪β防御素-1(Porcineβdefensin 1,PBD-1)的基因导入该细胞,然后将稳定整合外源基因的细胞移植到母鼠乳腺组织;利用宿主乳腺组织的微环境,诱导移植的细胞在体内进一步分化为乳腺相关细胞。结果如下:
(1)从昆明白小鼠超排获得的囊胚中挑选出560枚质量较好的胚胎,分别采用以下两类方式培养:①以不同传代数(0～5代)的小鼠胎儿成纤维细胞(Mouseembryonic fibroblast,MEF)作饲养层培养小鼠胚胎;②添加条件培养基并以第3代MEF作饲养层培养胚胎。比较发现,在第0、1、2、3～5代的饲养层上培养小鼠胚胎,其ICM形成率及类ES细胞的最高传代数无明显差别。以ES细胞上清液条件培养基培养小鼠胚胎,无论是ICM的形成率,还是类ES细胞最高传代数等指标上,都明显优于普通ES细胞培养基。利用RT-PCR检测到ES细胞的特异表达基因Oct-4。ES细胞经AKP染色呈阳性,周围饲养层细胞和分化细胞不染色或轻染。ES细胞特异性抗体Oct-4及SSEA-1免疫荧光反应也呈阳性。此外,体内、体外分化试验发现所分离的昆明白小鼠类ES细胞在体内、体外均具有分化为多种类型细胞的潜能且细胞核型正常,证实其发育的多能性,可以作为后续研究的试验材料。
(2)将第3代小鼠类ES细胞集落用玻璃针撕成小块(每块含50～100个细胞)接种于MEF饲养层上连续培养10～13 d不传代,在MEF细胞分泌的细胞因子的作用下,小鼠类ES细胞定向分化为上皮样细胞,经过长期传代、纯化培养,建立了稳定的细胞系(目前该细胞系保存于中国典型培养物保藏中心(China Center forType Culture Collection,CCTCC))。RT-PCR检测结果表明,不同传代数小鼠类ES细胞来源的上皮样细胞(第13代和第23代)均不表达Oct-4,其他上皮细胞特异表达基因(K10,K14,K18,K19和Involucrin)在表达量上有一定的差异,但E-cadherin表达差异不明显。为进一步量化不同阶段分化细胞的基因表达水平的差异,用荧光定量PCR对筛选的上皮细胞不同阶段特异表达基因(K14、K19、Involucrin和E-cadherin)进行了定量分析,结果表明,不同代数的上皮样分化细胞(P13和P23)之间,K14和K19两个上皮细胞阶段特异表达基因的mRNA水平发生变化极显著(P＜0.01),前者水平增加到7倍,后者下降到原来的十分之一。而Involucrin和E-cadherin的表达水平差异不显著(P＞0.05)。经免疫细胞化学染色,广谱角蛋白(PCK)和另外两种上皮细胞特异蛋白标记(K14和K18)均在昆明白小鼠类ES细胞来源的上皮样分化细胞的胞浆着色,流式分析术进一步证实,表达K14、K18和PCK的细胞量分别为51.3%、67.4%和82.7%。表明分化细胞为上皮类型细胞。细胞增殖检测结果表明,80%左右的细胞处于活跃增殖期。综合以上结果得出结论:小鼠类ES细胞来源的上皮样细胞实质为上皮前体细胞。
(3)采用PCR方法从从猪肝脏组织RNA扩增得到252 bp的PBD-1编码区域,然后从质粒pcDNA3.1-BLG-HNP-1中获取了大小为879 bp的山羊β-乳球蛋白基因(BLG)片段,以其作为乳蛋白基因调控序列,构建了山羊乳腺特异性表达载体pIRES2-EGFP-BLG-PBD-1。经PCR鉴定、限制性内切酶酶切分析和克隆片段序列测定、比较和鉴定。证实成功构建了乳腺特异性表达载体pIRES2-EGFP-BLG-PBD-1,为进一步研究PBD-1蛋白的抗菌活性、抗菌机理及将进一步该载体应用于动物乳腺生物反应器的研究奠定基础。
(4)利用脂质体介导的方法,将山羊乳腺特异性表达载体pIRES2-EGFP-BLG-PBD-1质粒导入小鼠类ES细胞来源的上皮前体细胞,经过长期高浓度G418筛选,最终建立了稳定转染猪的β防御素-1(porcineβdefensin 1,PBD-1)基因的细胞系。利用半定量RT-PCR方法进一步分析,发现稳定转染后的细胞依然表达上皮特异表达基因E-cadherin、K18、K19、K14和K10(其中K14、K18是上皮细胞发育过程早期出现的标记基因),不表达多能干细胞特异表达基因Oct-4,也不表达上皮细胞晚期标记基因Invoclurin和成纤维细胞特异表达基因FSP1。这些基因的表达模式与细胞转染前相一致。免疫细胞化学鉴定,也证实稳定转染后细胞中上皮细胞特异标记蛋白PCK、K14和K18仍然有很高的表达水平。
(5)将稳定整合外源基因PBD-1的小鼠类ES细胞来源的上皮前体细胞以不同剂量(10~7个细胞/对乳腺和5×10~3个细胞/对乳腺)移植到不同发育阶段的同品系母鼠第四对乳腺组织内,通过荧光显微镜检测荧光标记(EGFP),证实移植后2周,仅能在高剂量细胞移植组观察到移植细胞的生存,而在移植后5周,只有妊娠中期和妊娠晚期的高剂量细胞移植组可以观察到生存下来的移植细胞,且经PCR检测到外源基因PBD-1的表达。小鼠类ES细胞来源的转基因上皮前体细胞在小鼠乳腺组织微环境的诱导下,进一步分化为CALLA~+或EMA~+阳性细胞(EMA和CALLA分别作为管腔细胞和肌上皮细胞特异的表面标记,用于分离这两种类型的细胞可以达到98%的纯度)。统计结果显示,5周后的分化效果明显优于2周,CALLA~+或EMA~+阳性细胞占生存细胞总数的比例分别达到28.4%和41.7%。此外还发现,移植5周后,受体母鼠乳腺组织经HE染色,与相同发育阶段的未移植昆明白母鼠比较,形态上无明显变化。表明细胞移植后乳腺组织发育正常。
(6)利用转基因上皮前体细胞移植策略成功建立了小鼠乳腺生物反应器模型。8只受体母鼠移植转基因上皮前体细胞2周后,其中3只(37.5%)母鼠乳汁中检测到外源蛋白PBD-1的表达,表达水平达到514.09±11.47 ng/mL。Western blotting分析得到一条7 ku左右的特异条带,与预期表达的蛋白分子量大小一致,进一步证实移植转基因mES-dK前体细胞2周后,受体母鼠乳汁中成功表达出了目的蛋白PBD-1。
ES cell technology has been the greatest way to product animal mammary gland bioreactor immediately since the first ES cell line was established in 1981. However, ES or ES-like cell line of livestock is difficult to generate and the culture technology which meets the requirement of product animal mammary gland bioreactor could hardly be achieved. In addition, nowadays, the predominant method to generate animal mammary gland bioreactor is to make transgenic animals, which is of great difficulty to implement in practical experiments, and it is definitely a waste of time, money and energy. Therefore, the extension and dissipation of this technique is extremely limited. It is especially important to search a new way to generate animal mammary gland bioreactor quickly.
Directed differentiation in vitro serves as one of the most popular research areas in stem cells for decades. Theoretically, more than 200 types of cells could be differentiated from Embryonic Stem (ES) cells in vitro. To date, many types of cells including neural precursor cells, islet cell, keratinocyte, epithelial precursor cell, myocardial cell and germocyte, have been derived from ES cells. It was reported that these ES derived precursor cell or stem cell can further differentiate into receipt related type of cells under microenvironmental induction in vivo after they were transplanted into some special tissue or organ, and survived, integrated in vivo and generated normal physiological function. In addition, some literatures reported that, gene-modified mammary epithelial cells can survive for long time in vivo and expressed foreign gene after they were transplanted into receipt breast tissue. In summary, these results indicated that, it is feasible to produce animal mammary gland bioreactor by cell transplantation of mES-like derived transgenic precursor cells.
Consequently, we propose a hypothesis with a model animal of mouse. We suppose that after stably transfected with a mammary-specific expression plasmid containing beta-lactoglobulin (BLG) gene promoter or whey acidic protein (WAP) orβ-casein, the mES derived epithelial precursor cells were transplanted into the mammary gland tissue, if the foreign cells would have survived, integrated in the recipient mammary gland and played normal physiological function there, the interest protein should be secreted into milk by mammary gland, that is, animal mammary gland bioreactor was generated.
In this research, mES (at passage three) derived epithelial precursor cells stably transfected with a mammary-specific expression plasmid containing beta-lactoglobulin (BLG) gene promoter and foreign gene PBD-1 (porcineβdefensin 1) were transplanted into the mammary gland tissue during pregnancy. These cells can survive and integrate in the recipient mammary gland under the microenvironmental induction and further differentiate into mammary related type of cells. The main results are as follows:
1. 560 collected 3.5- to 4-d old embryos were cultured in DMEM as follows. Method one: MEF (Mouse embryonic fibroblast) cells at different passages (from passage 0 to 5) were used as feeder cells. Method two: the third passage of MEF cells were used as feeder cells and cultured embryos with conditioned medium. On day 5, ICM (Inner cell mass)-outgrowth were isolated from blastocysts and plated on new feeder cells. Three to five days later, these cultures from ICMs have a high ratio of nucleus to cytoplasm, prominent nucleoli, and typical colony with ES cells' morphological characteristics. They have three dimensional prominent, round or elliptic colonies, with smooth and strongly refractive edges, and have no clear boundaries of cells within a colony. When two methods were compared, Method two is significantly better than Method one on the form ratio of ICM and passage number. These cells were subsequently characterized by RT-PCR and found to be positive for mouse pluripotent embryonic stem cell specific markers Oct-4. In addition, these cells expressed alkaline phosphatase, and staining intensity for SSEA-1 and Oct-4 in brown color while feeder layer failed to stain as control. These cells were diploid chromosome number, and they can differentiate into many types of cells in vitro and in vivo. This validated the pluripotent of mES-like cells obtained in our experiments. Therefore, they can be used as test material later.
2. To initiate differentiation, mES-like cell colonies (passage 3) were dispersed to small clumps, containing approximately 50 to 100 cells, thus allowed to grow on mitotically inactivated MEF feeder cells. For the additional 10-13 d of culture, the epithelial-like polygonal cells appeared around the disc-like colonies. They migrated and spreaded until cells were confluent with a typical paving stone-like appearance. These cells were called mES-derived keratinocytes (mES-dK) in this experiment. After the first six passages of co-culture with MEF feeder layers, mES-dK cells were plated onto a new flask without MEF feeder layers. The mES-dK cell line was generated and preservated in CCTCC (China Center for Type Culture Collection). RT-PCR results indicated that, K14, K18, K10, Involucrin and E-cadherin were found to be up regulated in the cells during the differentiation, whereas Oct-4 and K19 were down regulated (compared to pre-differentiation levels). To further assess the expression level during the differentiational process, we also analyzed the expression patterns of K14, K19, Involucrin, and Ecadherin by quantitative real-time PCR during the course of differentiation of mES cells into keratinocyte-like cells. The K14 transcript increased dramatically within 50 d (from mES cells to passage 13) after further differentiation in mES cells committed by removing MEF feeder layers. This level rose to about seven-fold (from passage 13 to 23). On the other hand, expression of K19 RNA rose dramatically during the initial phase (from mES cells to passage 13) and dropped back to a lower level (from passage 13 to 23). It is interesting to note that the expression patterns of Involucrin and E-cadherin transcripts both stayed at the similar level between 13 and 23 passages. Immunocytochemistry showed that almost all mES-dK cells were stained with pCK, K14, and K18, which are epithelial cell surface markers. To check if the keratinocyte-like cells were proliferating, we analyzed the cells by flowing cytometry for BrdU-incorporation and for keratins (K14, K18, and pCK) expression, the majority of cells are positive in both cases (80.5%, 51.3%, 67.4% and 82.7%, respectively). Thus we conclude that the epithelial-like cells are proliferating and presumed it may be an epithelial progenitor lineage.
3. The whole cDNA sequence encoding PBD-1 which was amplified by PCR from hepatic cDNA of porcine. BLG gene was amplified from the vector of pcDNA3.1-BLG-HNP-1 and inserted into a eukaryotic expression plasmid pIRES2-EGFP-PBD-1. The recombinant colonies were identified by the methods of restriction enzyme digestion, PCR and sequencing. These results showed that, the mammary gland special expression vector pIRES2-EGFP-BLG-PBD-1 was successfully constructed. The establishment of mammary gland special expression vector pIRES2-EGFP-BLG-PBD-1 serve as a foundation in research on antimicrobial activities and its mechanism of the defensins, and it plays a pivotal role in further research for the establishment of animal mammary bioreactor.
4. The pIRES2-EGFP-BLG-PBD-1 plasmid was transfected into mES-dK cells by Lipofectamine 2000. Stably transfected clones were selected using G418 at 2000μg/mL for first 5 weeks and surviving cells were maintained in 1000μg/mL G418. RT-PCR results indicated that, the transgenic mES-dK precursor cells expressed foreign genes PBD-1 and BLG, and the epithelial cell markers, K10, K14, K18 and K19, but did not express Oct-4, Invoclurin or Fibroblast special protein 1(FSP1). Immunocytochemistry showed that majority of mES-dK cells were stained with pCK, K14, and K18, which are used to mark epithelial cell surface.
5. Transgenic mES-dK cells were transplanted at different dose into inguinal #4 glands of Kunming white mice. Each animal received a 0.1ml injection of either vehicle (medium) alone or suspension of 5×10~3 or 1×10~7 transgenic mES-dK cells. Survival cells were mainly found in high dose cell graft groups two weeks after transplantation, and five weeks after operation, cells were only found in mid-pregnant group and late-pregnant group with the high dose cell graft. RT-PCR results indicated that, foreign gene PBD-1 was expressed in transplanted mammary gland tissue. Transgenic mES-dK cells further differentiated into EMA~+ or CALLA~+ cells, which were the exclusive surface markers of luminal and myoepithelial cells, respectively, and can be used to separate the cell types to the degree of 98% purity, and the largest percentage of EMA-positive grafted cell was 41.7% and CALLA-positive grafted cell was 28.4% at the 5-week survival period, respectively. In order to determine whether there were some changes on the structure of mammary gland after transplantation with mES-dK cells, HE staining of mammary gland was performed simultaneously. However, there was not significant difference about morphological characteristics of the mammary gland before and after the transplantation.
6. We produced a mouse model of animal mammary bioreactor by precursor cell transplantation of transgenic mES-dK cells. Two weeks later, 37.5% (3/8) female transplanted mice had PBD-1 expression in their milk. The pBD-1 concentration was measured with an ELISA kit and the PBD-1 contents in milk reached 514.09±11.47 ng/mL. Western blotting analysis further confirmed that PBD-1 protein (7 ku) was secreted in the milk 2 weeks after transplantation.
体外定向分化的研究一直是干细胞领域研究的热点,理论上ES细胞可以分化为200多种类型的细胞,目前已经获得了ES细胞来源的神经前体细胞、胰岛细胞、角质细胞、上皮前体细胞、心肌细胞、生殖细胞等多种类型的细胞。研究发现,将这些ES细胞来源的前体/干细胞移植后可以在宿主体内微环境诱导下进一步分化为特定细胞,并与周围组织整合,发挥正常生理功能。经过基因修饰后的乳腺上皮细胞被移植到宿主乳腺组织后可长期生存并表达外源基因。因此,利用ES细胞来源的前体/干细胞移植策略结合基因修饰技术构建动物乳腺生物反应器是可行的:以小鼠为动物模型,将稳定转染目的基因(由山羊乳球蛋白基因(β-lactoglobulin,BLG)调控的)的小鼠类ES细胞来源的上皮前体细胞移植到同源小鼠的乳腺组织,随着移植细胞在宿主乳腺组织生存、整合并发挥正常的生理功能,受体动物的乳腺可以分泌含有目的蛋白的乳汁,最终达到快速建立乳腺生物反应器模型的目的。
本研究将昆明白小鼠早期类ES细胞在体外定向分化为上皮前体细胞,通过稳定转染的方式,将携带编码猪β防御素-1(Porcineβdefensin 1,PBD-1)的基因导入该细胞,然后将稳定整合外源基因的细胞移植到母鼠乳腺组织;利用宿主乳腺组织的微环境,诱导移植的细胞在体内进一步分化为乳腺相关细胞。结果如下:
(1)从昆明白小鼠超排获得的囊胚中挑选出560枚质量较好的胚胎,分别采用以下两类方式培养:①以不同传代数(0～5代)的小鼠胎儿成纤维细胞(Mouseembryonic fibroblast,MEF)作饲养层培养小鼠胚胎;②添加条件培养基并以第3代MEF作饲养层培养胚胎。比较发现,在第0、1、2、3～5代的饲养层上培养小鼠胚胎,其ICM形成率及类ES细胞的最高传代数无明显差别。以ES细胞上清液条件培养基培养小鼠胚胎,无论是ICM的形成率,还是类ES细胞最高传代数等指标上,都明显优于普通ES细胞培养基。利用RT-PCR检测到ES细胞的特异表达基因Oct-4。ES细胞经AKP染色呈阳性,周围饲养层细胞和分化细胞不染色或轻染。ES细胞特异性抗体Oct-4及SSEA-1免疫荧光反应也呈阳性。此外,体内、体外分化试验发现所分离的昆明白小鼠类ES细胞在体内、体外均具有分化为多种类型细胞的潜能且细胞核型正常,证实其发育的多能性,可以作为后续研究的试验材料。
(2)将第3代小鼠类ES细胞集落用玻璃针撕成小块(每块含50～100个细胞)接种于MEF饲养层上连续培养10～13 d不传代,在MEF细胞分泌的细胞因子的作用下,小鼠类ES细胞定向分化为上皮样细胞,经过长期传代、纯化培养,建立了稳定的细胞系(目前该细胞系保存于中国典型培养物保藏中心(China Center forType Culture Collection,CCTCC))。RT-PCR检测结果表明,不同传代数小鼠类ES细胞来源的上皮样细胞(第13代和第23代)均不表达Oct-4,其他上皮细胞特异表达基因(K10,K14,K18,K19和Involucrin)在表达量上有一定的差异,但E-cadherin表达差异不明显。为进一步量化不同阶段分化细胞的基因表达水平的差异,用荧光定量PCR对筛选的上皮细胞不同阶段特异表达基因(K14、K19、Involucrin和E-cadherin)进行了定量分析,结果表明,不同代数的上皮样分化细胞(P13和P23)之间,K14和K19两个上皮细胞阶段特异表达基因的mRNA水平发生变化极显著(P＜0.01),前者水平增加到7倍,后者下降到原来的十分之一。而Involucrin和E-cadherin的表达水平差异不显著(P＞0.05)。经免疫细胞化学染色,广谱角蛋白(PCK)和另外两种上皮细胞特异蛋白标记(K14和K18)均在昆明白小鼠类ES细胞来源的上皮样分化细胞的胞浆着色,流式分析术进一步证实,表达K14、K18和PCK的细胞量分别为51.3%、67.4%和82.7%。表明分化细胞为上皮类型细胞。细胞增殖检测结果表明,80%左右的细胞处于活跃增殖期。综合以上结果得出结论:小鼠类ES细胞来源的上皮样细胞实质为上皮前体细胞。
(3)采用PCR方法从从猪肝脏组织RNA扩增得到252 bp的PBD-1编码区域,然后从质粒pcDNA3.1-BLG-HNP-1中获取了大小为879 bp的山羊β-乳球蛋白基因(BLG)片段,以其作为乳蛋白基因调控序列,构建了山羊乳腺特异性表达载体pIRES2-EGFP-BLG-PBD-1。经PCR鉴定、限制性内切酶酶切分析和克隆片段序列测定、比较和鉴定。证实成功构建了乳腺特异性表达载体pIRES2-EGFP-BLG-PBD-1,为进一步研究PBD-1蛋白的抗菌活性、抗菌机理及将进一步该载体应用于动物乳腺生物反应器的研究奠定基础。
(4)利用脂质体介导的方法,将山羊乳腺特异性表达载体pIRES2-EGFP-BLG-PBD-1质粒导入小鼠类ES细胞来源的上皮前体细胞,经过长期高浓度G418筛选,最终建立了稳定转染猪的β防御素-1(porcineβdefensin 1,PBD-1)基因的细胞系。利用半定量RT-PCR方法进一步分析,发现稳定转染后的细胞依然表达上皮特异表达基因E-cadherin、K18、K19、K14和K10(其中K14、K18是上皮细胞发育过程早期出现的标记基因),不表达多能干细胞特异表达基因Oct-4,也不表达上皮细胞晚期标记基因Invoclurin和成纤维细胞特异表达基因FSP1。这些基因的表达模式与细胞转染前相一致。免疫细胞化学鉴定,也证实稳定转染后细胞中上皮细胞特异标记蛋白PCK、K14和K18仍然有很高的表达水平。
(5)将稳定整合外源基因PBD-1的小鼠类ES细胞来源的上皮前体细胞以不同剂量(10~7个细胞/对乳腺和5×10~3个细胞/对乳腺)移植到不同发育阶段的同品系母鼠第四对乳腺组织内,通过荧光显微镜检测荧光标记(EGFP),证实移植后2周,仅能在高剂量细胞移植组观察到移植细胞的生存,而在移植后5周,只有妊娠中期和妊娠晚期的高剂量细胞移植组可以观察到生存下来的移植细胞,且经PCR检测到外源基因PBD-1的表达。小鼠类ES细胞来源的转基因上皮前体细胞在小鼠乳腺组织微环境的诱导下,进一步分化为CALLA~+或EMA~+阳性细胞(EMA和CALLA分别作为管腔细胞和肌上皮细胞特异的表面标记,用于分离这两种类型的细胞可以达到98%的纯度)。统计结果显示,5周后的分化效果明显优于2周,CALLA~+或EMA~+阳性细胞占生存细胞总数的比例分别达到28.4%和41.7%。此外还发现,移植5周后,受体母鼠乳腺组织经HE染色,与相同发育阶段的未移植昆明白母鼠比较,形态上无明显变化。表明细胞移植后乳腺组织发育正常。
(6)利用转基因上皮前体细胞移植策略成功建立了小鼠乳腺生物反应器模型。8只受体母鼠移植转基因上皮前体细胞2周后,其中3只(37.5%)母鼠乳汁中检测到外源蛋白PBD-1的表达,表达水平达到514.09±11.47 ng/mL。Western blotting分析得到一条7 ku左右的特异条带,与预期表达的蛋白分子量大小一致,进一步证实移植转基因mES-dK前体细胞2周后,受体母鼠乳汁中成功表达出了目的蛋白PBD-1。
ES cell technology has been the greatest way to product animal mammary gland bioreactor immediately since the first ES cell line was established in 1981. However, ES or ES-like cell line of livestock is difficult to generate and the culture technology which meets the requirement of product animal mammary gland bioreactor could hardly be achieved. In addition, nowadays, the predominant method to generate animal mammary gland bioreactor is to make transgenic animals, which is of great difficulty to implement in practical experiments, and it is definitely a waste of time, money and energy. Therefore, the extension and dissipation of this technique is extremely limited. It is especially important to search a new way to generate animal mammary gland bioreactor quickly.
Directed differentiation in vitro serves as one of the most popular research areas in stem cells for decades. Theoretically, more than 200 types of cells could be differentiated from Embryonic Stem (ES) cells in vitro. To date, many types of cells including neural precursor cells, islet cell, keratinocyte, epithelial precursor cell, myocardial cell and germocyte, have been derived from ES cells. It was reported that these ES derived precursor cell or stem cell can further differentiate into receipt related type of cells under microenvironmental induction in vivo after they were transplanted into some special tissue or organ, and survived, integrated in vivo and generated normal physiological function. In addition, some literatures reported that, gene-modified mammary epithelial cells can survive for long time in vivo and expressed foreign gene after they were transplanted into receipt breast tissue. In summary, these results indicated that, it is feasible to produce animal mammary gland bioreactor by cell transplantation of mES-like derived transgenic precursor cells.
Consequently, we propose a hypothesis with a model animal of mouse. We suppose that after stably transfected with a mammary-specific expression plasmid containing beta-lactoglobulin (BLG) gene promoter or whey acidic protein (WAP) orβ-casein, the mES derived epithelial precursor cells were transplanted into the mammary gland tissue, if the foreign cells would have survived, integrated in the recipient mammary gland and played normal physiological function there, the interest protein should be secreted into milk by mammary gland, that is, animal mammary gland bioreactor was generated.
In this research, mES (at passage three) derived epithelial precursor cells stably transfected with a mammary-specific expression plasmid containing beta-lactoglobulin (BLG) gene promoter and foreign gene PBD-1 (porcineβdefensin 1) were transplanted into the mammary gland tissue during pregnancy. These cells can survive and integrate in the recipient mammary gland under the microenvironmental induction and further differentiate into mammary related type of cells. The main results are as follows:
1. 560 collected 3.5- to 4-d old embryos were cultured in DMEM as follows. Method one: MEF (Mouse embryonic fibroblast) cells at different passages (from passage 0 to 5) were used as feeder cells. Method two: the third passage of MEF cells were used as feeder cells and cultured embryos with conditioned medium. On day 5, ICM (Inner cell mass)-outgrowth were isolated from blastocysts and plated on new feeder cells. Three to five days later, these cultures from ICMs have a high ratio of nucleus to cytoplasm, prominent nucleoli, and typical colony with ES cells' morphological characteristics. They have three dimensional prominent, round or elliptic colonies, with smooth and strongly refractive edges, and have no clear boundaries of cells within a colony. When two methods were compared, Method two is significantly better than Method one on the form ratio of ICM and passage number. These cells were subsequently characterized by RT-PCR and found to be positive for mouse pluripotent embryonic stem cell specific markers Oct-4. In addition, these cells expressed alkaline phosphatase, and staining intensity for SSEA-1 and Oct-4 in brown color while feeder layer failed to stain as control. These cells were diploid chromosome number, and they can differentiate into many types of cells in vitro and in vivo. This validated the pluripotent of mES-like cells obtained in our experiments. Therefore, they can be used as test material later.
2. To initiate differentiation, mES-like cell colonies (passage 3) were dispersed to small clumps, containing approximately 50 to 100 cells, thus allowed to grow on mitotically inactivated MEF feeder cells. For the additional 10-13 d of culture, the epithelial-like polygonal cells appeared around the disc-like colonies. They migrated and spreaded until cells were confluent with a typical paving stone-like appearance. These cells were called mES-derived keratinocytes (mES-dK) in this experiment. After the first six passages of co-culture with MEF feeder layers, mES-dK cells were plated onto a new flask without MEF feeder layers. The mES-dK cell line was generated and preservated in CCTCC (China Center for Type Culture Collection). RT-PCR results indicated that, K14, K18, K10, Involucrin and E-cadherin were found to be up regulated in the cells during the differentiation, whereas Oct-4 and K19 were down regulated (compared to pre-differentiation levels). To further assess the expression level during the differentiational process, we also analyzed the expression patterns of K14, K19, Involucrin, and Ecadherin by quantitative real-time PCR during the course of differentiation of mES cells into keratinocyte-like cells. The K14 transcript increased dramatically within 50 d (from mES cells to passage 13) after further differentiation in mES cells committed by removing MEF feeder layers. This level rose to about seven-fold (from passage 13 to 23). On the other hand, expression of K19 RNA rose dramatically during the initial phase (from mES cells to passage 13) and dropped back to a lower level (from passage 13 to 23). It is interesting to note that the expression patterns of Involucrin and E-cadherin transcripts both stayed at the similar level between 13 and 23 passages. Immunocytochemistry showed that almost all mES-dK cells were stained with pCK, K14, and K18, which are epithelial cell surface markers. To check if the keratinocyte-like cells were proliferating, we analyzed the cells by flowing cytometry for BrdU-incorporation and for keratins (K14, K18, and pCK) expression, the majority of cells are positive in both cases (80.5%, 51.3%, 67.4% and 82.7%, respectively). Thus we conclude that the epithelial-like cells are proliferating and presumed it may be an epithelial progenitor lineage.
3. The whole cDNA sequence encoding PBD-1 which was amplified by PCR from hepatic cDNA of porcine. BLG gene was amplified from the vector of pcDNA3.1-BLG-HNP-1 and inserted into a eukaryotic expression plasmid pIRES2-EGFP-PBD-1. The recombinant colonies were identified by the methods of restriction enzyme digestion, PCR and sequencing. These results showed that, the mammary gland special expression vector pIRES2-EGFP-BLG-PBD-1 was successfully constructed. The establishment of mammary gland special expression vector pIRES2-EGFP-BLG-PBD-1 serve as a foundation in research on antimicrobial activities and its mechanism of the defensins, and it plays a pivotal role in further research for the establishment of animal mammary bioreactor.
4. The pIRES2-EGFP-BLG-PBD-1 plasmid was transfected into mES-dK cells by Lipofectamine 2000. Stably transfected clones were selected using G418 at 2000μg/mL for first 5 weeks and surviving cells were maintained in 1000μg/mL G418. RT-PCR results indicated that, the transgenic mES-dK precursor cells expressed foreign genes PBD-1 and BLG, and the epithelial cell markers, K10, K14, K18 and K19, but did not express Oct-4, Invoclurin or Fibroblast special protein 1(FSP1). Immunocytochemistry showed that majority of mES-dK cells were stained with pCK, K14, and K18, which are used to mark epithelial cell surface.
5. Transgenic mES-dK cells were transplanted at different dose into inguinal #4 glands of Kunming white mice. Each animal received a 0.1ml injection of either vehicle (medium) alone or suspension of 5×10~3 or 1×10~7 transgenic mES-dK cells. Survival cells were mainly found in high dose cell graft groups two weeks after transplantation, and five weeks after operation, cells were only found in mid-pregnant group and late-pregnant group with the high dose cell graft. RT-PCR results indicated that, foreign gene PBD-1 was expressed in transplanted mammary gland tissue. Transgenic mES-dK cells further differentiated into EMA~+ or CALLA~+ cells, which were the exclusive surface markers of luminal and myoepithelial cells, respectively, and can be used to separate the cell types to the degree of 98% purity, and the largest percentage of EMA-positive grafted cell was 41.7% and CALLA-positive grafted cell was 28.4% at the 5-week survival period, respectively. In order to determine whether there were some changes on the structure of mammary gland after transplantation with mES-dK cells, HE staining of mammary gland was performed simultaneously. However, there was not significant difference about morphological characteristics of the mammary gland before and after the transplantation.
6. We produced a mouse model of animal mammary bioreactor by precursor cell transplantation of transgenic mES-dK cells. Two weeks later, 37.5% (3/8) female transplanted mice had PBD-1 expression in their milk. The pBD-1 concentration was measured with an ELISA kit and the PBD-1 contents in milk reached 514.09±11.47 ng/mL. Western blotting analysis further confirmed that PBD-1 protein (7 ku) was secreted in the milk 2 weeks after transplantation.
引文
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