Relevance of spontaneous activity to urinary bladder function: an in vitro and in vivo study

The presence and functional significance (if any) of spontaneous activity in the normal urinary bladder during filling is a controversial subject. One model used by many investigators to study spontaneous activity has been isolated urinary bladder smooth muscle strips. Although spontaneous activity is a property commonly observed in isolated urinary bladder strip preparations, the in vitro whole bladder preparation (rabbit) is devoid of spontaneous activity. Additionally, under normal conditions the in vivo rabbit bladder does not display spontaneous activity during the filling phases of micturition. The present study compares the spontaneous activity of isolated smooth muscle strips, the whole bladder preparation, and the catheterized in vivo bladder (rabbit). The results are as follows: The spontaneous activity (frequency and amplitude) of isolated strips is extremely variable among strips of the same bladder. Spontaneous activity is not affected by the following specific inhibitory compounds: tetrodotoxin, atropine, phentolamine, propranolol and hexamethonium. This indicates that spontaneous activity observed in isolated strips is myogenic in nature and not dependent on the activation of specific autonomic receptors. The in vitro whole bladder preparation shows no spontaneous activity at any volume or pressure unless longitudinal tension is applied. The spontaneous activity of the whole bladder subjected to longitudinal tension is not affected by the same compounds mentioned above. Spontaneous activity of the in vivo bladder is absent at low intravesical volumes and pressures. Spontaneous activity develops upon reaching a critical pressure. However, this activity is completely inhibited by intravenous ganglionic blockade (hexamethonium). In the presence of hexamethonium, the in vivo bladder is devoid of spontaneous activity at any volume or pressure, thus the in vivo "spontaneous activity" is mediated through neuronal reflexes. It is concluded that under normal circumstances the rabbit bladder is devoid of myogenic spontaneous activity and that the spontaneous activity observed in isolated strips is directly related to longitudinal stretch. Since under normal conditions the bladder is not subjected to longitudinal stretch, the spontaneous activity observed in the isolated strip studies has little physiological significance under normal conditions, but could help explain the pathophysiology of certain dysfunctions during the filling stage of micturition.     

In vitro and in vivo effect of verapamil on human airway responsiveness to leukotriene D4.

The mechanism by which leukotriene D4 (LTD4) induces airway narrowing in man is unclear. We have investigated this by examining the effect of the calcium channel blocker verapamil on the sensitivity of in vitro preparations of human bronchi to LTD4 and methacholine, and on the bronchoconstriction induced in normal subjects by these agonists in vivo. In vitro smooth muscle sensitivity was assessed by the concentration of LTD4 and methacholine causing a 50% of maximum contraction (EC50) and as the maximum tension generated. Verapamil did not alter baseline tension or the response to LTD4 but did inhibit contractile responses to methacholine. In vivo studies were performed in six normal subjects; they inhaled increasing concentrations of LTD4 (0.4-50 micrograms/ml) or methacholine (2-64 mg/ml). Airway responsiveness in vivo was expressed as the provocation concentration (PC) of agonist producing a 35% fall in specific airways conductance (PC35sGaw) and a 30% fall in flow at 30% of vital capacity (PC30 V30(p)). Verapamil did not alter baseline sGaw or V30(p). One subject did not respond to LTD4 on either day. In contrast to the in vitro results, verapamil produced a greater than 10 fold reduction in LTD4 induced bronchoconstriction, but had no effect on methacholine induced bronchoconstriction. These results suggest that in normal subjects bronchoconstriction induced by inhaled LTD4 is due to a combination of direct and indirect mechanisms.     

Microencapsulation of human parathyroid cells: an "in vitro" study

BACKGROUND: Patients affected by hypoparathyroidism of variable etiology are currently treated with exogenously administered vitamin D and calcium. Human parathyroid transplantation has long been investigated as a possible mean of treating these patients to prevent long-term hypocalcemia. However, the main obstacle for this treatment is represented by tissue rejection. A reliable method to efficiently protect the transplanted tissue from rejection and to allow long-term survival of the graft is the encapsulation of tissues or cells in alginate-polylysine-alginate membranes, which were successfully used for encapsulation of islets of Langerhans. The microencapsulation of parathyroid tissue fragments or of parathyroid cells becomes, therefore, a potential approach for the successful treatment of permanent symptomatic hypoparathyroidism without pharmacological immunosuppression. MATERIALS AND METHODS: We describe microencapsulation of differentiated human parathyroid cells derived from adenoma or hyperplastic glands. Long-term viability, cell growth, and parathyroid hormone production of microencapsulated cells were evaluated together with responsiveness to extracellular Ca(2+). RESULTS: Microencapsulated parathyroid cells maintained proliferative and differentiative properties for a long term in culture with a good response to extracellular Ca(2+) concentration. CONCLUSIONS: These findings represent a crucial step toward the construction of functional bioartificial parathyroid organoids for the treatment of hypoparathyroidism in humans.     

Proliferation and differentiation of human tenocytes in response to platelet rich plasma: an in vitro and in vivo study

Platelet rich plasma (PRP) is the autologous plasma fraction with a platelet-rich cellular component which is enriched with a number of growth factors. Due to its availability and low cost, PRP has become an increasingly popular clinical tool as an alternative source of growth factors for various applications, for example, tendon regeneration but with limited success in clinical trials. The main objective of the current study was to determine whether activated PRP [i.e., platelet rich plasma-clot release (PRCR)] could be used to induce the proliferation and collagen synthesis in human tenocyte in vitro. The advantage of using PRCR is that the platelet-derived bioactive factors are more concentrated and could initiate a more rapid and accelerated healing response than PRP. Our results demonstrated that 10% PRCR treatment accelerated the extent of cell proliferation and collagen production by human tenocytes in vitro. The expression of specific tenocyte markers were similar to conventional fetal bovine serum (FBS)-treated tenocytes implanted in mice within 14 days of implantation in diffusion chambers. Moreover, relatively more collagen fibrils were evident in PRCR-treated tenocytes in vivo as compared to 10% FBS-treated cells. Overall, our feasibility study has indicated that PRCR can induce human tenocyte proliferation and collagen synthesis which could be implemented for future tendon regeneration in reconstructive surgeries.     

Turnover of human tubular cells exposed to proteins in vivo and in vitro

BACKGROUND: The cause of tubulointerstitial pathology in glomerular disease is uncertain. Proteinuria may be a causative factor and has been shown to increase the turnover of tubular cells in a rat model of proteinuria. We investigated whether increased tubular cell proliferation occurs in human proteinuric renal disease. A human cell culture model was used to investigate the effects of proteins on tubular cell turnover further. METHODS: Tubular proliferation in renal biopsies from patients with membranous nephropathy (MN) and minimal change nephropathy (MCN) was assessed by in situ hybridization for histone mRNA. Proliferation was measured in polarized human tubular cell cultures using tritiated thymidine, following addition of proteins to the apical medium. Toxicity was assessed by lactate dehydrogenase (LDH) release and monolayer permeability to inulin. RESULTS: Increased tubular cell histone mRNA expression occurred in biopsies in MN (3.0-fold increase, P < 0.002) and MCN (3.6-fold increase, P < 0.02). Serum proteins added to the medium on human tubular cell cultures increased thymidine uptake (1.3-fold, P < 0.005), LDH release (1.5-fold, P < 0.001), and monolayer permeability (1.7-fold, P < 0.005). The effects were reproduced by a fraction of molecular weight 40 to 100 kD, but not by pure albumin or transferrin. CONCLUSIONS: Proliferation of tubular cells is associated with proteinuria in vivo and is caused by proteins in cell culture. Toxicity of proteins to tubular cells and increased cell turnover may contribute to tubulointerstitial pathology in glomerular disease.     

Synergism between BCNU and irradiation in the treatment of anaplastic gliomas. An in vivo study using the avian sarcoma virus-induced glioma model.

The therapeutic effects of irradiation, BCNU, or combined irradiation and BCNU were studied in the avian sarrcoma virus (ASV)-induced glioma model in rats. Whole-head orthovoltage radiation therapy was given in six equal fractions over 2 weeks, and BCNU was administered intraperitoneally as a single dose of 10 mg/kg. Two series of experiments were performed in order to duplicate the results. In Series I, the median survival times of the experimental groups, in days after randomization were as follows: control group (no treatment), 69; group receiving 200 rads, 84 (p less than 0.05); group receiving BCNU, 80.5 (p less than 0.1); and group receiving 2000 rads + BCNU, 112 (p less than 0.001). In Series 2, the median survival times were: control group, 73.5; group receiving 2300 rads, 85 (p less than 0.01); group receiving BCNU, 92.5 (p less than 0.025); and group receiving 2300 rads + BCNU, 123.5 (p less than 0.001). In both series, combined therapy was significantly better than either radiation or BCNU alone. This is the first time that a synergistic effect of BCNU and irradiation has been reported in an in vivo brain-tumor model and supports the clinical use of this combination in the treatment of malignant gliomas.     

Laser photochemotherapy of rhodamine-123 sensitized human glioma cells in vitro

The photochemotherapeutic effect of the mitochondria-specific dye rhodamine-123 (Rh-123) on human glioma cells in culture was studied. Cultured U-251MG glioma cells were incubated for 30 minutes in 10 micrograms/ml of Rh-123 and then exposed to blue-green light between 488 and 514.5 nm using a continuous-wave argon laser. Cells that were treated with Rh-123 and the argon laser at power densities less than 200 mW/sq cm demonstrated increasing tumor-cell killing with increasing time of exposure to laser light. Tumor-cell killing achieved with power densities of light less than 200 mW/sq cm was shown to be due solely to a photochemical effect and not to a direct (thermal) effect of the laser. The photochemical effect was dependent upon the intracellular concentration of Rh-123 and the length of light exposure, and not the intensity of light. The selective retention of Rh-123 by glioma cells and its exclusion from normal cells in conjunction with its photoactivated cytotoxicity suggest that Rh-123 may be a useful photosensitizing drug for the treatment of malignant gliomas in situ.     

体内外人脂肪间充质干细胞向肝样细胞分化的实验研究

目的 分离、培养人脂肪间充质干细胞(hADSCs),研究其在体内外向类肝样细胞转化的能力.方法 采用Ⅰ型胶原酶消化皮下脂肪,贴壁分选去除造血细胞的方法获得较纯的干细胞.通过对形态观察、表面分子鉴定、透射电镜内部结构扫描以及成脂、成骨定向诱导对脂肪间充质干细胞进行鉴定.用EGF、FGF、OSM、HGF、TSA细胞因子体外诱导hADSCs向肝样细胞分化.尾静脉输注hADSCs至急性肝损伤裸鼠,观察hADSCs在裸鼠肝脏内定植、向肝样细胞分化情况,以及hADSCs对裸鼠肝损伤的保护作用.结果 分离获得的细胞呈长梭形贴壁生长,低表达CD34、CD45,高表达CD73、CD90、CD105,具有多向分化潜能.诱导细胞表现出肝样细胞形态,表达AFP、alb,并且和肝细胞一样具有吸脂性.体内诱导:移植细胞在裸鼠体内生存、分布,并且细胞表达人alb,细胞体积较裸鼠肝脏细胞大,形态不均一,与人肝脏细胞形态有差异.同时研究发现hADSCs可改善急性肝损伤裸鼠肝功能,降低转氨酶.结论 人脂肪间充质干细胞可在体内外向肝样细胞分化,分化的细胞具有肝细胞样生物学性状.hADSCs对裸鼠急性肝损伤具有一定的治疗作用.

Abstract：

Objective To isolate and culture human adipose derived mesenchynal stem cells (hADSCs) and study the potential of hADSCs to differentiate into hepatocyte-like cells. Methods To ensure the removal of contaminating hematopoietic cells, hADSCs were selected based on plastic adherence. Cell surface antigen was confirmed by flow cytometry; ultramicrostructure was detected by transmission electron microscopy; adipogenic and osteogenic differentiation of hADSCs was analyzed by oil Red O staining and yon Kossa staining. hADSCs were exposed to differentiation medium containing EGF,FGF,OSM, HGF,TSA in vitro. 10% CCl4 (100 μ1/20 g body weight )was injected into immune-deficient BALB/c-nu mice and hADSCs (5 × 105 cells) were simultaneously administrated from the tail vein. Blood samples were collected and concentration of aminotransferase and direct bilirubin were detected. Administration without hADSCs was used as a control. One month later, we sacrificed the mice and liver sections were examined by histochemical immunofluorescence with human ALB specific antibodies. Results hADSCs exhibited fibroblast-like morphology, and expressed CD73, CD90,CD105, and were lacking of CD34 and CD45. Adipogenic and osteogenic differentiation showed that hADSCs have the capacity of multidifferentiation. The differentiated cells showed hepatocyte-like cell morphologies and hepatocyte-specific markers including albumin (alb) and α-fetoprotein (AFP). The bioactivity assays revealed that these hepatocyte-like cells could uptake low-density lipoprotein (LDL).Histochemical immunofluorescence showed that hADSCs were incorporated into injured livers. Some human alb-positive cells were found in liver sections after implantation of undifferentiated hADSCs. Transaminase activity in the experimental group was lower than in the control group. Conclution hADSCs can differentiated into functional hepatocyte-like cells and can relieve CCl4 induced BALB/c-nu acute liver injury.     

Proliferation,migration, and invasion of human glioma cells exposed to fractionated radiotherapy in vitro

Radiotherapy is a well established treatment for malignant gliomas. This study describes the migration, proliferation, and invasion behaviour of two human glioma cell lines (GaMg and U-87 Mg) grown as multicellular tumour spheroids after radiotherapy. Migration and proliferation studies were performed using conventional and accelerated fractionation up to 60 Gy and 59.4 Gy, respectively. A dose-dependent growth and migratory response to irradiation independent of the type of fractionation was observed. A coculture system in which tumour spheroids were confronted with foetal rat brain aggregates was used for invasion studies. Marked invasion of the glioma spheroids into the brain aggregates occurred with or without radiotherapy. For the GaMg cells, flow cytometric DNA histograms after treatment with 10 Gy and 40 Gy showed an accumulation of cells in the G2/M phase of the cell cycle. Radiotherapy inhibits tumour cell growth and migration, but the invasiveness of the remaining tumour cells seems to be unaffected.     

Proliferation,migration, and invasion of human glioma cells exposed to fractionated radiotherapy in vitro

Radiotherapy is a well established treatment for malignant gliomas. This study describes the migration, proliferation, and invasion behaviour of two human glioma cell lines (GaMg and U-87 Mg) grown as multicellular tumour spheroids after radiotherapy. Migration and proliferation studies were performed using conventional and accelerated fractionation up to 60 Gy and 59.4 Gy, respectively. A dose-dependent growth and migratory response to irradiation independent of the type of fractionation was observed. A coculture system in which tumour spheroids were confronted with foetal rat brain aggregates was used for invasion studies. Marked invasion of the glioma spheroids into the brain aggregates occurred with or without radiotherapy. For the GaMg cells, flow cytometric DNA histograms after treatment with 10 Gy and 40 Gy showed an accumulation of cells in the G2/M phase of the cell cycle. Radiotherapy inhibits tumour cell growth and migration, but the invasiveness of the remaining tumour cells seems to be unaffected.     

体内外人脂肪间充质干细胞向肝样细胞分化的实验研究

Objective To isolate and culture human adipose derived mesenchynal stem cells (hADSCs) and study the potential of hADSCs to differentiate into hepatocyte-like cells. Methods To ensure the removal of contaminating hematopoietic cells, hADSCs were selected based on plastic adherence. Cell surface antigen was confirmed by flow cytometry; ultramicrostructure was detected by transmission electron microscopy; adipogenic and osteogenic differentiation of hADSCs was analyzed by oil Red O staining and yon Kossa staining. hADSCs were exposed to differentiation medium containing EGF,FGF,OSM, HGF,TSA in vitro. 10% CCl4 (100 μ1/20 g body weight )was injected into immune-deficient BALB/c-nu mice and hADSCs (5 × 105 cells) were simultaneously administrated from the tail vein. Blood samples were collected and concentration of aminotransferase and direct bilirubin were detected. Administration without hADSCs was used as a control. One month later, we sacrificed the mice and liver sections were examined by histochemical immunofluorescence with human ALB specific antibodies. Results hADSCs exhibited fibroblast-like morphology, and expressed CD73, CD90,CD105, and were lacking of CD34 and CD45. Adipogenic and osteogenic differentiation showed that hADSCs have the capacity of multidifferentiation. The differentiated cells showed hepatocyte-like cell morphologies and hepatocyte-specific markers including albumin (alb) and α-fetoprotein (AFP). The bioactivity assays revealed that these hepatocyte-like cells could uptake low-density lipoprotein (LDL).Histochemical immunofluorescence showed that hADSCs were incorporated into injured livers. Some human alb-positive cells were found in liver sections after implantation of undifferentiated hADSCs. Transaminase activity in the experimental group was lower than in the control group. Conclution hADSCs can differentiated into functional hepatocyte-like cells and can relieve CCl4 induced BALB/c-nu acute liver injury.     

Generation of male germ cells from induced pluripotent stem cells (iPS cells): an in vitro and in vivo study

Recent studies have reported that induced pluripotent stem (iPS) cells from mice and humans can differentiate into primordial germ cells. However, whether iPS cells are capable of producing male germ cells is not known. The objective of this study was to investigate the differentiation potential of mouse iPS cells into spermatogonial stem cells and late-stage male germ cells. We used an approach that combines in vitro differentiation and in vivo transplantation. Embryoid bodies (EBs) were obtained from iPS cells using leukaemia inhibitor factor (LIF)-free medium. Quantitative PCR revealed a decrease in Oct4 expression and an increase in Stra8 and Vasa mRNA in the EBs derived from iPS cells. iPS cell-derived EBs were induced by retinoic acid to differentiate into spermatogonial stem cells (SSCs), as evidenced by their expression of VASA, as well as CDH1 and GFRα1, which are markers of SSCs. Furthermore, these germ cells derived from iPS cells were transplanted into recipient testes of mice that had been pre-treated with busulfan. Notably, iPS cell-derived SSCs were able to differentiate into male germ cells ranging from spermatogonia to round spermatids, as shown by VASA and SCP3 expression. This study demonstrates that iPS cells have the potential to differentiate into late-stage male germ cells. The derivation of male germ cells from iPS cells has potential applications in the treatment of male infertility and provides a model for uncovering the molecular mechanisms underlying male germ cell development.     

Influence of different strategies of volume replacement on the activity of matrix metalloproteinases: an in vitro and in vivo study

BACKGROUND: Excessive production of matrix metalloproteinase 9 (MMP-9) is linked to tissue damage and anastomotic leakage after large bowel surgery. Hence, the aim of this study was to verify whether different strategies of fluids administration can reduce MMP-9 expression. METHODS: In the in vitro experiment, the authors tested the hypothesis of a direct inhibition of MMP-9 by the fluids used perioperatively, i.e., lactated Ringer's solution, 3.4% poligeline, and hydroxyethyl starch 130/0.4. In the in vivo experiment, 36 patients undergoing surgery for colon cancer were randomly assigned to three groups to receive lactated Ringer's solution, poligeline, or hydroxyethyl starch. MMP-9 and tissue inhibitor of metalloproteinases were measured from venous blood samples; the MMP-9/tissue inhibitor of metalloproteinases ratio was calculated as an index of equilibrium between the action of MMP-9 and its inhibition. RESULTS: In the in vitro experiment, the presence of hydroxyethyl starch 130/0.4 in the MMP-9 assay system showed a strong inhibition of the enzymatic activity compared with lactated Ringer's solution. In the in vivo experiment, MMP-9 and tissue inhibitor of metalloproteinases plasma levels did not differ among the three groups at baseline, whereas those levels increased significantly at the end of surgery. At that time, the MMP-9 plasma levels and the MMP-9/tissue inhibitor of metalloproteinases ratio were significantly higher in the lactated Ringer's solution and poligeline groups than in the hydroxyethyl starch group. These results were confirmed 72 h after surgery. CONCLUSIONS: This study demonstrates that hydroxyethyl starch 130/04 decreases the circulating levels of MMP-9 in patients undergoing abdominal surgery.     

A modified culture system for epidermal cells for grafting purposes: an in vitro and in vivo study

A fully differentiated epithelium mimicking the features of native epidermis was obtained in vitro by culturing human or porcine epidermal keratinocytes on polyester filter substrate at the air-liquid interface. In addition, after 2 weeks of culture, hemidesmosome-like structures were formed along the basal area of the plasma membrane of the basal cells at the cell-filter interface. When grafted onto full-thickness skin wounds in pigs, the take of cell sheets detached from the filter with dispase was significantly higher (about 70%) in comparison to mechanically detached keratinocytes (about 15%). With dispase-treated keratinocytes alone, basement membrane formation took place within 7 days postgrafting as judged from the presence of a lamina lucida and positive staining for type IV collagen. Also, numerous hemidesmosomes and anchoring fibrils were observed at the basal cell-"neodermis" interface. The fully differentiated epidermis, generated by culturing keratinocytes at the air-liquid interface and detached from the substrate by dispase-treatment, is less fragile and easier to handle than epidermal autografts obtained by conventional culturing methods. Detachment by a short dispase-treatment appeared in our hands the only method for successful and complete epithelial regeneration in full-thickness wounds.