The induction of new ductal growth in adult prostatic epithelium in response to an embryonic prostatic inductor

Tissue recombinants were prepared with a single epithelial ductal tip from adult prostate and mesenchyme from either the embryonic urogenital sinus or adult urinary bladder. Recombinants were grown in vivo beneath the renal capsule of male hosts. After 4 weeks of in vivo growth, extensive growth of arborizing ducts was apparent in recombinants composed of urogenital sinus mesenchyme and a single adult prostatic ductal tip. One-dimensional polyacrylamide gel electrophoresis indicated that these recombinants contained many of the proteins of the mature prostate. Heterospecific recombinants (rat urogenital sinus mesenchyme and mouse prostatic epithelium) showed the ductal tissue to be derived solely from the prostatic epithelium. In recombinants of a prostatic ductal tip with mesenchyme from the urinary bladder, ductal growth was absent, the ductal tip was maintained as a single, discrete, epithelial structure, and the protein composition of these recombinants more closely resembled that of the bladder. The results demonstrate that the epithelial cells of the adult prostate can participate in new ductal growth in response to an embryonic prostatic inductor. These data provide experimental evidence to support the hypothesis that human benign prostatic hyperplasia may result from the anomalous reactivation of embryonic growth potential in the adult prostate.     

Prostate stem cells and benign prostatic hyperplasia

Pharmacological approaches are available to medically-managed patients with symptomatic BPH before surgical intervention is required. These include daily treatment with alpha-blockers and 5-alpha-reductase inhibitors alone or in combination. These medical approaches have two major problems. First, treatments are chronic and must be taken daily. Second, there are significant financial costs and quality of life issues for such chronic treatments. Is it possible to develop effective acute therapy for symptomatic BPH without the long-term androgen deprivation-induced side effects? Two seminal but rarely cited studies of Walsh [Peters, Walsh: N Engl J Med 317:599-604, 1987] and Coffey et al. [Sufrin et al.: Invest Urol 13:418-423, 1976], combined with the growing understanding of the stem cell organization of the prostate stromal (S) and epithelial (E) compartments and their reciprocal paracrine and autocrine interactions provides the rationale for an acute approach.The Walsh study documents that: (1) androgen deprivation disrupts the reciprocal interaction between the prostate S and E thereby decreasing the weight of both compartments and (2) once BPH develops, androgen deprivation does not decrease the number of stem cell units in either the S or E compartments since subsequent androgen restoration fully restores the enlarged gland. The Coffey study documents that acute androgen deprivation sensitizes S-E interactions to radiation induced disruptions so that following radiation, androgen restoration does not induce full gland regrowth. Therefore, effective therapy for symptomatic BPH should be achievable by acute treatment with reversible androgen deprivation for a limited period followed by a single dose of conformal external beam radiation before allowing the man to recovery his normal serum testosterone.     

Phosphotyrosine phosphatase activity of human and canine acid phosphatases of prostatic origin

Human and canine prostatic specimens containing high levels of acid phosphatase (AP) activity were tested, at acid pH, for their ability to hydrolyze the major phosphoaminoacids present in phosphorylated proteins, phosphoserine (p-ser), phosphothreonine (p-thr), and phosphotyrosine (p-tyr). The cleavage of a synthetic substrate, para-nitrophenyl-phosphate (p-npp), was also measured as an indicator of AP activity; its inhibition by sodium-L-tartrate (T) was used as a criterion to identify prostatic acid phosphatase (PAP). It was found that: 1) the Km of p-tyr and p-npp were 2.0 mM and 0.41 mM, respectively, with similar Vmax values (0.078 and 0.087 mumoles of phosphate (Pi) liberated per minute per milligram of protein); 2) the ID50 were 0.25 mM and 0.50 mM with sodium orthovanadate (VO4) and T, respectively, using p-npp as substrate-with p-tyr as substrate, the values obtained were 0.016 mM and 0.11 mM, respectively; 3) activity toward p-ser and p-thr was minimal; 4) native PAP from dog seminal plasma, with a molecular weight of 90-100 kD, as determined by gel filtration on HPLC, hydrolyzed p-tyr preferentially, and this phosphatase (Pase) activity was also strongly inhibited by both T and VO4; and 5) the AP present in human and canine prostatic tissue and cells, as well as in their secretions, also preferentially hydrolyzed phosphotyrosine, and it was inhibited by T and VO4. It is proposed that these p-tyr Pases may be involved in the local regulation of prostatic growth.     

Investigations of prostate epithelial stem cells and prostate cancer stem cells

Although both prostate epithelial stem cells and prostate cancer stem cells are implicated in the differentiation of the normal prostate gland and carcinogenesis of prostate cancer, there has, until recently, been little information regarding their biology. This review summarizes the recent advancements in cell biological research including various in vitro culture systems that have offered the characterization and isolation of prostate epithelial stem cells and prostate cancer stem cells. In addition, the stromal niche or microenvironment of stem cells plays an essential role in proliferation and differentiation of normal stem cells. Stroma surrounding cancer cells, which also provide another unique niche, may involve the initiation and development of cancer stem cells. Investigation of stem cells and their microenvironments in the prostate should lead to the elucidation of biological features and the development of novel treatments for prostate cancer.     

Serum and prostatic growth-promoting factors for steroid-independent epithelial cells of adult dog prostate

A growth factor-like effect has been observed on canine prostatic epithelial cells when cultured in the presence of their homologous serum and prostatic extracts; the mitogenic activities of both preparations were dose-dependent and not altered by charcoal treatment. The effect of dog serum decreased when the density of the epithelial cell cultures increased and was minimal on canine prostatic fibroblasts. Trace amounts of intracellular sex steroids did not contribute to epithelial cell proliferation since the presence of sex steroid action inhibitors did not alter growth rate; in those conditions, cycloheximide completely prevented cell division. When various hormones and known mitogenic agents were tested alone or in combination with steroids, none elicited an increase in the number of epithelial cells cultured in serum-free medium or altered the proliferative effect of dog serum observed in parallel cultures. On gel filtration, dog serum or tissue cytosol showed a major mitogenic activity at an apparent molecular mass of 150 kDa and a minor one of 1.5 kDa as evaluated by gel filtration of dog serum ultrafiltrate. Acidic extraction of prostatic tissue followed by chromatography on a hydrophobic C-18 column and subsequent gel filtration also led to the detection of the low Mr component. Thus, humoral and/or tissular factors present in vivo and different from known mitogens may be of importance as direct modulators of the basal epithelial cell growth in the adult canine prostate.     

A study of oligosaccharide determinants expressed by prostatic glandular epithelium of the normal adult rat

Summary Normal prostates from Copenhagen/Fischer F1 hybrid rats were removed at 14 months of age. After routine formalin fixation and paraffin embedding, the expression of seven oligosaccharide structures by prostatic epithelial cells was assessed by an examination of lectin binding sites before and after neuraminidase digestion. Con-A bound to plasma membranes as well as the cytoplasm of all cells, thus confirming the presence of complex-type glycoconjugates. However, only two other oligosaccharides, apart from Con-A, were freely expressed on epithelial luminal plasma membranes. These were the Type I structure (Ga113GalNAc-) identified by PNA-binding and (GlcNAc14GlcNAc14-)_n identified by WGA. PNA, WGA, UEA-1 and SBA bound to the cytoplasm of almost all epithelial cells, although their intracellular distribution was not identical. DBF binding was not identified. ECG bound to only a very few cells and then only after digestion with neuraminidase when it was localised to the cytoplasm. Following removal of sialic acid groups by neuraminidase digestion, PNA-binding became more prominent, SBA-binding appeared localized to paranuclear intracellular vesicles and WGA binding sites were abolished. This study has now characterized the major oligosaccharide determinants expressed by rat normal prostatic epithelial cells and provides a baseline against which alterations occurring during ontogenesis and oncogenesis may be compared.Abbreviations Con-A Canavalia ensiformis - SBA Glycine max - DBF Dolichos biflorus - PNA Arachis hypogaea - ECG Erythrina cristagalli - WGA Triticum vulgaris - UEA-1 Ulex europaeus-1 This work was supported by a grant from the Hammersmith and Queen Charlotte's Special Health Authority     

人脂肪间充质干细胞向表皮细胞表型转化的研究

目的:探索人脂肪间充质干细胞(adipose derived mesenchymal stem cells,ADSCs)向表皮细胞表型转化的方法。方法:以手术中剩余的人皮下脂肪组织为材料来源,利用胶原酶消化法分离ADSCs,流式细胞仪检测细胞表面标记CD29、CD90、CD105的表达,成脂诱导后油红O染色鉴定。实验组利用第四军医大学西京医院烧伤与皮肤外科实验室已成功构建的pc DNA3.1(+)/SP+EGF质粒经脂质体介导转染的Ha Cat细胞,与ADSCs在Transwell小室中共培养,对照组为ADSCs加入10%FBS培养基,14天后Realtime-PCR检测两组ADSCs中CK19、integrin-β的m RNA表达量。结果:实验组ADSCs中CK19、integrin-β的m RNA表达量较对照组明显升高,且差别有统计学意义。结论:转染EGF的Ha Cat细胞可诱导ADSCs向表皮细胞表型分化,从而为其成为组织工程理想的种子细胞提供了进一步的支持。     

Cell separation and characterization of epithelial cells from human benign prostatic hyperplasia

Epithelial cells were isolated from human prostatic hyperplasia (BPH) after mechanical disruption of the tissue. The tissue was cut into small pieces, then mechanical pressure was applied. Stroma and epithelium were separated by a sequence of sedimentation steps. The recovery rate of epithelial cells was around 20 million cells per gram of tissue and more than 95% of the cells did exclude trypan blue. Epithelial cells can be identified by phase contrast microscopy and by the acid phosphatase content of the cells. In more than 95% of the cells the presence of acid phosphatase could be shown cytochemically with phosphorylcholine as a substrate. This finding indicates that the contamination with other cell types is very small. Histological study of the remaining stroma indicates that most of the epithelial cells are removed. Also, the acid phosphatase content of this fraction was found to be very low. The problem of obtaining large quantities of stromal cells in suspension has not yet been resolved. However, the technique described may be more suitable than others for the separate study of stroma and epithelium from BPH.     

Growth responses of normal, benign hyperplastic, and malignant human prostatic epithelial cells in vitro to cholera toxin, pituitary extract, and hydrocortisone

Normal, benign hyperplastic (BPH), and malignant prostatic epithelial cells from adult humans can be serially passaged in medium PFMR-4 supplemented with 20% whole serum, cholera toxin, and epidermal growth factor (EGF). Improved growth of these cells has been achieved by optimizing the concentrations of the components of the basal medium. In the new modified medium, PFMR-4A, the only supplements required are 5% dialyzed serum, cholera toxin, EGF, pituitary extract, and hydrocortisone. Comparative studies of the growth responses of prostatic cells derived from normal central zone, normal peripheral zone, BPH nodules, and adenocarcinomas did not reveal any qualitative differences; all respond positively to the addition of cholera toxin, pituitary extract, and hydrocortisone to the culture medium. Cells of one cancer strain, however, were much less density-dependent for growth than were the other strains of normal, BPH, or malignant cells.     

Requirements for attachment and subsequent growth of canine prostatic epithelial cells in culture

The attachment and spreading of canine prostatic epithelial cells in primary monolayers and their subsequent proliferation were studied in Primaria and in polystyrene dishes either uncoated or coated with collagen type I, fibronectin, laminin, poly-D-lysine, or natural extracellular matrices (nECM) produced by canine prostatic epithelial or fibroblastic cells. Cells were inoculated in serum-free medium or in medium supplemented with either dialyzed fetal bovine serum (dFBS) or charcoal-treated dog serum at 10%. Dihydrotestosterone (DHT) and 5 alpha-androstane 3 alpha, 17 beta-diol (3 alpha, 17 beta-diol) at 10(-6) M or a mixture of steroids (androstenedione, testosterone, DHT, 3 alpha, 17 beta-diol, 5 alpha-androstane 3 beta, 17 beta-diol, estrone, and estradiol) were also added. Of all components and dishes tested, only dFBS and the nECM produced by prostatic epithelial cells increased cell attachment (850% and 450%, respectively). When the latter preparations were used in combination, an additive effect (1,500%) was observed, and the subsequent addition of dog serum after the attachment period yielded the highest number of growing prostatic epithelial cells. When prostatic epithelial cells were inoculated either in dishes coated with a nECM derived from prostatic fibroblasts or in presence of dog serum, a 50% inhibition in their plating efficiency was observed. The presence of collagen type I, fibronectin, laminin, or an nECM in the cultures, together with various steroids, had no effect on the cell unresponsiveness to steroids nor did it alter the mitogenic effect of dog serum. Thus, the attachment and spreading of canine prostatic epithelial cells in monolayers are mediated by nonsteroidal factors present in dFBS and in their nECM. Their presence, together with steroids, does not elicit a proliferative response to steroids nor did it increase the effect of growth-promoting factors in dog serum.     

Characterization of rat ventral prostatic epithelial cells in collagen gel culture

The characterization of rat ventral prostatic epithelial cells grown in collagen gel culture was undertaken to determine the usefulness of this culture technique for the study of prostatic growth and differentiation in vitro. The results of these studies demonstrate that embedding prostatic epithelial cells in a matrix of collagen gel permitted rapid proliferation and maintenance of the cells for a prolonged period of time in a state that showed remarkable structural resemblance and physiological responsiveness to that found in vivo. Three-dimensional outgrowths from dissociated single epithelial cells and/or small aggregates of epithelial cells were obtained which resembled structures actually found within the prostate gland, as determined by histological and visual examination of the cellular growths present within the gels. In addition, the responsiveness of the cells in the collagen gel cultures to various hormonal additives, including insulin, testosterone, prolactin, and estradiol correlated closely to the manner in which these cells have been shown to respond in vivo. The general characteristics of growth of the cells in the collagen gel cultures, the effect of various sera and different pH levels upon growth, and the ability of the cells to produce acid phosphatase were also investigated.     

Nonsteroidal serum factors involved in the regulation of the proliferation of canine prostatic epithelial cells in culture

Canine prostatic epithelial cells were cultured in primary monolayers in order to define those factors that induce a proliferative response at the cellular level. Cultures were performed in a serum-free medium or in a medium supplemented either with fetal bovine serum or dog serum in the presence or absence of several sex steroids (androstenedione, testosterone, dihydrotestosterone, 3α- and 3β-androstanediols, epitestosterone, epidihydro-testosterone, estrone, estradiol, and progesterone). Cell proliferation was observed in the absence of serum and exogenous steroids. The rate of cell division was serum dependent and steroid independent. Pretreatment of sera with charcoal had no effect on their mitogenic activities. Cells maintained in an endocrine milieu prior to tissue dispersion and throughout the whole procedure proliferate to the same extent as those deprived of hormones, whether free of serum or added supplements. The addition of insulin (2 μg/ml), dog prolactin (up to 25 ng/ml) and zinc (10^?8 to 10^?2 M) in a serum-free medium did not induce cell responsiveness to steroids. Dihydrotestosterone, 3α-androstanediol, and estradiol alone or in combinations known to induce the growth of the canine prostate in vivo were ineffective in vitro. The proliferative responses to sera were time and concentration dependent, and dog serum was more potent than fetal bovine serum. Thus, humoral factors other than steroids, prolactin, insulin, or zinc may be of importance in the activation of epithelial cells involved in the development of prostatic hyperplasia and adenocarcinoma.     

TGF-β1和bFGFs体外诱导成人骨髓间充质干细胞表达软骨细胞表型的实验研究

目的：从人骨髓中分离间充质干细胞(mesenchymal stem cells，MSCs)，应用流式细胞学技术分析不同细胞因子对细胞增殖分化的影响并观察细胞组织化学特点。结果：MSCs具有独特的表征，即CD29阳性，CD34阴性：TGF—β1(transforming growth faetorsβ1，TGF—β1)、bFGFs(base fibroblast growth faethors，bFGFs)及两者的联合应用可诱导MSCs表达软骨细胞表型向成软骨细胞方向分化：结论：MSCs是一群均一的细胞，具有独特表征，本实验所采用的细胞分离方法为筛选合适的细胞体外扩增及分化提供了有意义的线索。     

肝细胞生长因子诱导骨髓间充质干细胞向肝细胞分化的实验研究

目的探讨成年大鼠骨髓间充质干细胞(MSCs)在体外是否可定向诱导分化为肝细胞及其方法。方法采用梯度离心法,分离纯化SD大鼠的MSCs,流式细胞仪检测和碱性磷酸酶染色鉴定细胞类型。根据培养基中肝细胞生长因子(HGF)浓度不同,将MSCs分为4组进行诱导分化：A组0 ng／ml,B组10ng／ml,C组20ng／ml,D组40ng／ml。倒置显微镜连续观察细胞分化过程的形态学变化。于培养的1,3,7,14,21,28 d,分别以逆转录聚合酶链反应(RT-PCR)和细胞免疫组化检测各组细胞甲胎蛋白(AFP)、细胞角蛋白18(CK18)和白蛋白的基因和蛋白表达。结果分离纯化的SD大鼠MSCs的表面标志为CD29^ ,CD44^ ,CD34^-,CD45^-和CD90^ ,细胞的碱性磷酸酶染色阴性,细胞纯度达98％以上。C组与D组的MSCs,于培养第7天出现AFP基因表达,第14天表达增强,第28天表达减弱;第14天始出现白蛋白、CK18基因表达,而后持续。B组和A组在培养过程中,未出现AFP、CK18和白蛋白的表达。C组与D组MSCs的AFP细胞免疫组化染色培养第7天即出现阳性,第14天白蛋白和CK18免疫组化染色阳性。A组和B组的MSCs的AFP、白蛋白和CK18的免疫组化染色均阴性。结论成年大鼠MSCs在较高浓度的HGF的诱导下,可分化为肝细胞。     

体外诱导骨髓间充质干细胞向肾小管上皮样细胞分化

Objective To investigate the differentiation of rat bone marrow mesenchymal stem cells (MSCs) to renal tubular epithelial-like cells under different conditions. Methods MSCs were obtained from rat marrow. MSCs were isolated by gradient density centrifugation and plastic adherence and then purified. Surface markers were identified with flow cytometry after amplification in vitro. The purified MSCs of the third passage were cultured respectively as follows: (1) control group: DMEM medium with fetal bovine serum(FBS). (2) all-trans retinoic acid (ATRA) group: DMEM medium with FBS, ATRA and ischemic reperfusion-injured kidney tissue homogenate. (3)combination group: DMEM medium with FBS, ATRA, ischemic reperfusion-injured kidney tissue homogenate, epidermal growth factor (EGF) and bone morphogenetic protein 7 (BMP-7). After 7 days, the MSCs were collected for alkaline phosphatase (AKP) staining, cytokeratin-18 and E-cadherin immunocytochemical analysis. Results The positive rates of the third passage MSCs in CD44, CD90 and CD29 were 97.8%±0.9%, 96.8%±1.4% and 97.6%±2.4%,respectively, but in CD11b/c and CD34 were only 13.2%±0.6% and 1.2%±0.5%. The MSCs in control group were spindle. The MSCs in ATRA group were round and elliptic. The MSCs in combination group became cobblestone-like cells after 7 days. AKP staining showed that tubular epithelial-like cells from MSCs in control group were negative, some above cells in ATRA group were positive and number of above cells increased in combination group. Compared with negative control group, the ratios of cytokeratin-18 positive cells in ATRA group and combination group were respectively increassed by 29.47%±1.08% and 47.52%±2.13% (all P<0.05), the ratios of E-cadherin positive cells in ATRA group and combination group were respectively increased by 14.88%±2.46% and 36.15%±1.13% (all P<0.05). Conclusion MSCs may differentiate by renal tubular epithelial-like cells under the induction of ischemic reperfusion-injured kidney tissue homogenate and ATRA in vitro, which are further differentiated under the combined induction of EGF and BMP-7.     

体外诱导骨髓间充质干细胞向肾小管上皮样细胞分化

Objective To investigate the differentiation of rat bone marrow mesenchymal stem cells (MSCs) to renal tubular epithelial-like cells under different conditions. Methods MSCs were obtained from rat marrow. MSCs were isolated by gradient density centrifugation and plastic adherence and then purified. Surface markers were identified with flow cytometry after amplification in vitro. The purified MSCs of the third passage were cultured respectively as follows: (1) control group: DMEM medium with fetal bovine serum(FBS). (2) all-trans retinoic acid (ATRA) group: DMEM medium with FBS, ATRA and ischemic reperfusion-injured kidney tissue homogenate. (3)combination group: DMEM medium with FBS, ATRA, ischemic reperfusion-injured kidney tissue homogenate, epidermal growth factor (EGF) and bone morphogenetic protein 7 (BMP-7). After 7 days, the MSCs were collected for alkaline phosphatase (AKP) staining, cytokeratin-18 and E-cadherin immunocytochemical analysis. Results The positive rates of the third passage MSCs in CD44, CD90 and CD29 were 97.8%±0.9%, 96.8%±1.4% and 97.6%±2.4%,respectively, but in CD11b/c and CD34 were only 13.2%±0.6% and 1.2%±0.5%. The MSCs in control group were spindle. The MSCs in ATRA group were round and elliptic. The MSCs in combination group became cobblestone-like cells after 7 days. AKP staining showed that tubular epithelial-like cells from MSCs in control group were negative, some above cells in ATRA group were positive and number of above cells increased in combination group. Compared with negative control group, the ratios of cytokeratin-18 positive cells in ATRA group and combination group were respectively increassed by 29.47%±1.08% and 47.52%±2.13% (all P<0.05), the ratios of E-cadherin positive cells in ATRA group and combination group were respectively increased by 14.88%±2.46% and 36.15%±1.13% (all P<0.05). Conclusion MSCs may differentiate by renal tubular epithelial-like cells under the induction of ischemic reperfusion-injured kidney tissue homogenate and ATRA in vitro, which are further differentiated under the combined induction of EGF and BMP-7.     

体外诱导骨髓间充质干细胞向肾小管上皮样细胞分化

Objective To investigate the differentiation of rat bone marrow mesenchymal stem cells (MSCs) to renal tubular epithelial-like cells under different conditions. Methods MSCs were obtained from rat marrow. MSCs were isolated by gradient density centrifugation and plastic adherence and then purified. Surface markers were identified with flow cytometry after amplification in vitro. The purified MSCs of the third passage were cultured respectively as follows: (1) control group: DMEM medium with fetal bovine serum(FBS). (2) all-trans retinoic acid (ATRA) group: DMEM medium with FBS, ATRA and ischemic reperfusion-injured kidney tissue homogenate. (3)combination group: DMEM medium with FBS, ATRA, ischemic reperfusion-injured kidney tissue homogenate, epidermal growth factor (EGF) and bone morphogenetic protein 7 (BMP-7). After 7 days, the MSCs were collected for alkaline phosphatase (AKP) staining, cytokeratin-18 and E-cadherin immunocytochemical analysis. Results The positive rates of the third passage MSCs in CD44, CD90 and CD29 were 97.8%±0.9%, 96.8%±1.4% and 97.6%±2.4%,respectively, but in CD11b/c and CD34 were only 13.2%±0.6% and 1.2%±0.5%. The MSCs in control group were spindle. The MSCs in ATRA group were round and elliptic. The MSCs in combination group became cobblestone-like cells after 7 days. AKP staining showed that tubular epithelial-like cells from MSCs in control group were negative, some above cells in ATRA group were positive and number of above cells increased in combination group. Compared with negative control group, the ratios of cytokeratin-18 positive cells in ATRA group and combination group were respectively increassed by 29.47%±1.08% and 47.52%±2.13% (all P<0.05), the ratios of E-cadherin positive cells in ATRA group and combination group were respectively increased by 14.88%±2.46% and 36.15%±1.13% (all P<0.05). Conclusion MSCs may differentiate by renal tubular epithelial-like cells under the induction of ischemic reperfusion-injured kidney tissue homogenate and ATRA in vitro, which are further differentiated under the combined induction of EGF and BMP-7.     

体外诱导骨髓间充质干细胞向肾小管上皮样细胞分化

Objective To investigate the differentiation of rat bone marrow mesenchymal stem cells (MSCs) to renal tubular epithelial-like cells under different conditions. Methods MSCs were obtained from rat marrow. MSCs were isolated by gradient density centrifugation and plastic adherence and then purified. Surface markers were identified with flow cytometry after amplification in vitro. The purified MSCs of the third passage were cultured respectively as follows: (1) control group: DMEM medium with fetal bovine serum(FBS). (2) all-trans retinoic acid (ATRA) group: DMEM medium with FBS, ATRA and ischemic reperfusion-injured kidney tissue homogenate. (3)combination group: DMEM medium with FBS, ATRA, ischemic reperfusion-injured kidney tissue homogenate, epidermal growth factor (EGF) and bone morphogenetic protein 7 (BMP-7). After 7 days, the MSCs were collected for alkaline phosphatase (AKP) staining, cytokeratin-18 and E-cadherin immunocytochemical analysis. Results The positive rates of the third passage MSCs in CD44, CD90 and CD29 were 97.8%±0.9%, 96.8%±1.4% and 97.6%±2.4%,respectively, but in CD11b/c and CD34 were only 13.2%±0.6% and 1.2%±0.5%. The MSCs in control group were spindle. The MSCs in ATRA group were round and elliptic. The MSCs in combination group became cobblestone-like cells after 7 days. AKP staining showed that tubular epithelial-like cells from MSCs in control group were negative, some above cells in ATRA group were positive and number of above cells increased in combination group. Compared with negative control group, the ratios of cytokeratin-18 positive cells in ATRA group and combination group were respectively increassed by 29.47%±1.08% and 47.52%±2.13% (all P<0.05), the ratios of E-cadherin positive cells in ATRA group and combination group were respectively increased by 14.88%±2.46% and 36.15%±1.13% (all P<0.05). Conclusion MSCs may differentiate by renal tubular epithelial-like cells under the induction of ischemic reperfusion-injured kidney tissue homogenate and ATRA in vitro, which are further differentiated under the combined induction of EGF and BMP-7.     

体外诱导骨髓间充质干细胞向肾小管上皮样细胞分化

Objective To investigate the differentiation of rat bone marrow mesenchymal stem cells (MSCs) to renal tubular epithelial-like cells under different conditions. Methods MSCs were obtained from rat marrow. MSCs were isolated by gradient density centrifugation and plastic adherence and then purified. Surface markers were identified with flow cytometry after amplification in vitro. The purified MSCs of the third passage were cultured respectively as follows: (1) control group: DMEM medium with fetal bovine serum(FBS). (2) all-trans retinoic acid (ATRA) group: DMEM medium with FBS, ATRA and ischemic reperfusion-injured kidney tissue homogenate. (3)combination group: DMEM medium with FBS, ATRA, ischemic reperfusion-injured kidney tissue homogenate, epidermal growth factor (EGF) and bone morphogenetic protein 7 (BMP-7). After 7 days, the MSCs were collected for alkaline phosphatase (AKP) staining, cytokeratin-18 and E-cadherin immunocytochemical analysis. Results The positive rates of the third passage MSCs in CD44, CD90 and CD29 were 97.8%±0.9%, 96.8%±1.4% and 97.6%±2.4%,respectively, but in CD11b/c and CD34 were only 13.2%±0.6% and 1.2%±0.5%. The MSCs in control group were spindle. The MSCs in ATRA group were round and elliptic. The MSCs in combination group became cobblestone-like cells after 7 days. AKP staining showed that tubular epithelial-like cells from MSCs in control group were negative, some above cells in ATRA group were positive and number of above cells increased in combination group. Compared with negative control group, the ratios of cytokeratin-18 positive cells in ATRA group and combination group were respectively increassed by 29.47%±1.08% and 47.52%±2.13% (all P<0.05), the ratios of E-cadherin positive cells in ATRA group and combination group were respectively increased by 14.88%±2.46% and 36.15%±1.13% (all P<0.05). Conclusion MSCs may differentiate by renal tubular epithelial-like cells under the induction of ischemic reperfusion-injured kidney tissue homogenate and ATRA in vitro, which are further differentiated under the combined induction of EGF and BMP-7.