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BACKGROUND:The use of mesenchymal stem cels in the field of tissue engineering for osteoarticular injury repair is a very promising tool since these cels are readily expandable and able to differentiate into chondrocytes. Abundant evidence suggests that microRNAs play critical roles in chondrogenic differentiation of mesenchymal stem cels. OBJECTIVE:To observe the chondrogenic effect of human bone marrow mesenchymal stem cels transfected with lentiviral vectors bearing miR-221-3p/222-3p inhibition, thereby provding new strategies for cartilage injury. METHODS: miRNA microarray technology was applied to detect microRNAs expression profiles at three different stages of chondrogenic differentiation induction after transforming growth factor-β3 treatment and verified by real-time fluorescence quantitative PCR (RT-qPCR). Human bone marrow mesenchymal stem cels were infected with lentivirus bearing miR-221-3p/222-3p inhibition. After co-suppressing the expression of miR-221/222-3p, cel counting kit-8 was used to determine the cel proliferation, the differentiation of bone marrow mesenchymal stem cels towards chondrocytes was verified by type II colagen protein expression through immunohistochemistry and glycosaminoglycan accumulation was also elevated by sarranine O staining. RT-PCR was used to detect type II colagen and aggrecan mRNA expression at 21 days of chondrogenic induction. RESULTS AND CONCLUSION: The expression of miR-221-3p/222-3p was inhibited after Lv-miR221-3p/222-3p inhibition co-transfected into bone marrow mesenchymal stem cels. microRNA microarray and RT-qPCR results showed that the expression of miR-221-3p/222-3p was declined significantly at the anaphase of chondrogenic differentiation. The expression levels of chondrogenic markers, Aggrecan and type II colagen were significantly increased in the miR-221-3p/222-3p inhibition group and cel proliferation was also inhibited significantly compared with non-transduced cels or transduced with the empty lentiviral vector group. miR-221-3p/222-3p knockdown in bone marrow mesenchymal stem cels could inhibit proliferation but promote chondrogenic differentiation of bone marrow mesenchymal stem cels.
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OBJECTIVE: Cisplatin is a widely used chemotherapeutic agent in the treatment of cancers in clinic; but it often induces adverse effects on ovarian functions such as reduced fertility and premature menopause. Mesna could attenuate the cisplatin-induced ovarian damages; however, the underlying mechanism is still unknown. This study aimed to figure out the underlying mechanism of the protection of mesna for ovaries against cisplatin therapy in cancers. METHODS: We performed female adult Sprague-Dawley rats into normal saline control (NS), low-dose cisplatin (CL), high-dose cisplatin (CH), CL plus mesna (CL+M), and CH plus mesna (CH+M) groups and detected anti-Mullerian hormone (AMH)-positive follicle, oxidative stress status and anti-oxidative capability in ovaries. RESULTS: AMH-positive follicles were significantly decreased after cisplatin administration, which was significantly reversed when mesna was co-administered with cisplatin. The end product of lipid peroxidation, malondialdehyde (MDA), was significantly increased, but the anti-oxidative enzymatic activity of superoxide dismutase (SOD) and glutathione (GSH) were significantly decreased in cisplatin groups when compared with NS group. In contrast, after co-administration of cisplatin with mesna, MDA was significantly decreased whereas the activity of SOD and the concentration of GSH were increased. Moreover, mesna did not decrease the anti-tumor property of cisplatin in HePG2 cell lines. CONCLUSION: Cisplatin damages the granulosa cells by oxidative stress to deplete the ovarian reserve and mesna could protect ovarian reserve through anti-oxidation. These results might highlight the mechanism of the protection of mesna for ovarian reserve and open an avenue for the application of mesna as a protective additive in cisplatin chemotherapy in clinical practise.
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Adult , Animals , Female , Humans , Rats , Anti-Mullerian Hormone , Cisplatin , Fertility , Glutathione , Granulosa Cells , Hep G2 Cells , Lipid Peroxidation , Malondialdehyde , Menopause, Premature , Mesna , Ovary , Oxidative Stress , Rats, Sprague-Dawley , Superoxide DismutaseABSTRACT
Objective To investigate the approaches for diagnosis and treatment of intrahepatic cholangiocarcinoma(ICC)and asgess its prognosis factors.Methods The clinical data of 86 patients with ICC in our hospital from January 1995 to December2005 were retrospeetively analyzed.All patients were divided into two groups according to the treatment method,including hepatectomy and lymphatic clearance group(Group A,n=42)and hepateetomy group(Group B,n=44),and their clinicopathological variables were analyzed.Resuits The 1-,3-and 5-year survival rates were 77.81%and 35.21%,20.93%and 19.82%,2.31%and 0%respectively between group A and group B.There was significantly difference between these two groups(P＜0.01).The analysis showed that resection and lymphatic clearance were correlated to prognosis.The 1-,3-and 5-year survival rates were 59.21%,26.21%,and 20.11% respectively in 47 patients who were found no lymph node metastasis,and the 1-,3-and 5-year survival rates were 19.82%,2.31%and 0% respectively in 39 patients who were found lymph node metastasis.There was significantly difference in survival rate between group A and group B(P＜0.01).Condusions Reseetability and lymphatic clearance are two significant factors correlated to survival of the patients with ICC.Aggresgive treatment of lymph node metastasis in hepatoduodenal ligament is an important strategy to improve survival rates and strengthen patient's life quality.
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Objective To investigate the distribution modes of NOS nerves and interstitial cells of Cajal (ICCs) in myenteric plexus of human fetal intestine. Methods Small intestine samples from 12 fetuses were collected for the preparation of whole mount preparations and cryo sections examined by immunocytochemistry for c kit receptor tyrosine kinase and histochemistry for NADPH diaphorase. Results ICCs associated with the myenteric plexus in the fetal small intestine were observed in the shape of spindle or ovoid with two to three slender processes forming an independent and complete cellular network. While NOS positive nerves, which constitute the main neuronal component of ganglia, connecting strands, and nerve fibers, were found within the circular muscle layer and myenteric plexus of the small intestine and the latter was especially rich of this kind of nerves. Most of these positive neurons were Dogiel I type neurons and they often gathered in cluster in the ganglia. Each ganglion contained several to dozens of NOS positive neurons. Although no colocalization of ICCs and NOS positive nerves was found by double staining of whole mount preparations and cryo sections, they were closely distributed. Conclusion Our results indicate that NO released by myenteric plexus, an inhibitory neurotransmitter, may possess the function of regulating ICCs and smooth muscle in late stage of the fetus.
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Objective To study the expression of estrogen receptor beta (ER ?) in the hypothalamic paraventricular nucleus (PVN) of maternal mice during pregnancy and lactation. Methods Nickel ammonium sulfate intensified immunocytochemical method was employed to investigate the expression of ER ? in the postnatal developing PVN of female mice. Results ER ? immunopositive materials were predominantly localized in the magnocellular division of PVN, and sparse positive cells were found in the parvocellular division. Most of the positive materials were found in the whole cell nuclei, but no obvious cytoplasma or process immunopositive cells were detected. In the pregnant female brain, generally, the ER ? level was lower. The lowest levels of expression were found at mid pregnancy, and then peaked at peripartum (from gestational days 18 to postpartum day 1), followed by another decrease to normal adult level from postpartum day 4. Conclusion The above results suggest that during pregnancy and lactation, ER ? may be predominantly involved in the PVN regulation of parturitional process.
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Objective To study the effect of hypoxia on the expression of interleukin 6 (IL 6) in cocultured pulmonary arterial smooth muscle cells (PASMC) Methods Rat PASMC were cocultured with rat lung microvascular endothelial cells, and randomly divided into 5 groups: normal group (N), 2 h hypoxia (H2), 6 h hypoxia (H6), 12 h hypoxia (H12), and 24 h hypoxia (H24) The expression level of IL 6 mRNA in PASMC was detected with RT PCR, and the activity of IL 6 in the supernatant with radioimmunoassay Results The expression level of IL 6 mRNA increased in PASMC in H2, reached the highest in H6, and decreased in H12, but still higher than that of N The changes of the IL 6 activity in the supernatant as well as IL 6 mRNA expression in the cells were in a time dependent manner Conclusion Hypoxia can enhance the expression of IL 6 in cocultured PASMC And it may activate others genes which regulate the hypoxic response of PASMC and signal transduction
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Objective To isolate and identify pancreatic stem cells of Kunming mouse and to observe the differentiation potency of those cells. Methods Pancreas of postnatal Kunming mice were digested by enzymes to isolate pancreatic stem cells. The potency of the cell differentiation was then identified with special marker of cytokeratin 19(CK 19), insulin and glucagons by immunocytochemical staining. Results Few epithelioid cells could be found to survive at the beginning of isolation, but when medium was replaced by that with growth factor, the cells proliferated quickly in fusiform shape and formed colony gradually. The cells were CK 19 immunoreactive positive after transfer of culture. After differentiation induced by high glucose, the cells formed the pancreatic islet like structures. Immunocytochemical staining showed that part of the cells of pancreatic islet like structures were insulin immunoreactive positive and few of the cells were glucagon immunoreactive positive. Conclusion Pancreatic stem cells of Kumming mouse can proliferate when cultured in vitro and have the potency of differentiation into ? and? cells of pancreatic islet.
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Objective To observe the changes of the gene expressions at spinal cord injury site of rat after transplantation of BDNF genetically modified neural stem cells(NSCs) so as to provide basic data for the repair of spinal cord injury. Methods The Wistar rats were randomly divided into 4 groups: control group, operation group, NSCs transplantation group, BDNF NSCs transplantation group. Four time points(7 day, 1 month, 2 month, 3 month) were divided for each group. The expressions of ? galactosidase and BDNF, GFAP, NF 200 at the site of spinal cord injury were observed by cell transplantation, X gal histochemistry, immunocytochemistry, in situ hybridization, etc. Results After transplantation of BDNF genetically modified NSCs, some X gal positive cells were found at the sections of spinal cord injury. The expressions of BDNF were strong, especially at 1 week and 1 month post transplantation in transplantation group. The GFAP and NF 200 positive cells were also found at each time point in each group. Conclusion BDNF genetically modified NSCs can survive at the site of spinal cord injury and can strongly express BDNF, suggesting that BDNF genetically modified NSCs can be used as the material for the repair of spinal cord injury.
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Objective To investigate the nerve recanalization and the motor function of hind legs after transplantation of BDNF genetically modified neural stem cells(NSCs) at spinal cord injury site in rat. Methods After L4 spinal cord transection of rat, BDNF genetically modified NSCs were transplanted immediately. Retrograde HRP tracing through sciatic nerve were practiced at 1 week, 1 month, 2 month, 3 month after transplantation of BDNF genetically modified NSCs. The morphological changes were observed at section of spinal cord and the motor functions of both hind legs of rat were detected. Results The morphology of the injured spinal cord sections turned better. Retrograde HRP tracing through sciatic nerve showed some HRP positive neurons and nerve fibers at the site of near rostral end of the nearly injured part at one month after transplantation and increased with the time going by. Motor function of hind legs of rats recovered significantly in all transplantation groups. Conclusion BDNF genetically modified NSCs have repairing effect on spinal cord injury in rat.
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This paper is to report the autonomic innervation of the gall bladder and extrahepatic bile ducts studied on whole mount stretch preparations and cryostat sections with acetylcholinesterase staining, glyoxylic acid induced fluorescence histochemical method, and PAP immunohistochemical method.The autonomic nerves of the gall bladder and extrahepatic bile dudts in cats consist of two plexuses, composed of cholinergic, adrenergic and peptidergic nerves, and situated in the subserosa and submucosa respectively. They are known as subserosal and submucosal ganglionated plexuses. They distribute in the region from the fundus of the bladder, to the neck and the cystic duct, through the common bile duct downward, finally to the duodenal papilla.The adrenergic nerves mainly run along the, blood vessels, distributing in the serosa and lamina propria of the mucosa of the bladder, and participating in the formation of the subepithelial and perivasctilar plexuses. The adrenergic nerves are most densely distributed in the cystic duct and mosl sparsely in the fundus. This finding is in good agreement with the tissue concentration of noradrenaline(NA) determined with biochemical asssay, which is also the highest in the tissue of the cystic duct. The difference of NA concentration between the cystic duct tissue and other biliary tissues was statistically Gignificant(P0.05).Immunohistochemical study revealed that there are 4 kinds of immunoactivc peptidergic nerves, i. e. nerves with vasoactive intestinal polypeptide (VIP), substance P(SP), L-enkephalin (L-ENK), and somatostatin (SOM) respectively. The 4 kinds of nerves distribute extensively in all the tissue layers of the ex-trahepatic biliary tract and participate in the formation of all the nerve plexuses. Among them, VIP nerves are the richest, then the SP and L-ENK nerves, and the SOM nerves the sparsest. In general, peptidergic nerves are densely distributed in the sphincter of Oddi.Finally the physiological significance of the peptidergic nerves was discussed.
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In this study gloxylic acid-induced fluorescent histochemical method was usedto reveal the innervation of adrenergic nerves,acetycholinesterase (AChE) methodto show the cholinergic nerves,and the immunohistochemical method to display thevasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) immunoreactivenerves.This observation on the vas deferens showed that the VIP-immunoreactivenerves had the same distribution as the cholinergic nerves,and mainly existed inlamina propria,while NPY-immunoreactive nerves had the same distribution asadrenergic nerves and mainly existed in the muscular layer.One group of animalswas treated by intraperitoneal injections of 6-OHDA on the lst,2nd,6th and 7thday,and killed on the 8th day.Following treatment with 6-OHDA,significantevanescence of most adrenergic nerves was observed.NPY-immunoreactive nervesreduced remarkably and the extent of the decrease was similiar to that of adrenergicnerves,whereas no change was observed on the VIP and cholinergic nerves.Theimmunoelectronic microscopy (PAP pre-embedding method) demonstrated that VIPimmunoreactive complex deposited in the varicosity containing small clear vesicles(diameter of 40-55nm).Some varicosities also contained small number of largegranular vesicles with the diameter of 100-144mm.NPY immunoreactive complexdeposited in the large granular vesicles,and occasionally can be observed in thesmall granular vesicles,40-55nm in diameter.The results from this study suggestthat VIP probably co-exist with ACh and NPY with noradrenalin in rat vasdeferens.
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SOM in sequence.The results of RIA wereconsistent with the density of the peptide-containing structures by ICC.
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By using the histochemical method, the electron microscopy and biochemical measurement, this study deals with the changes of the innervation of the uterus in tats induced by pregnancy. The results revealed that the adrenergic and cholinergic innervation of the uterus were reduced in advanced pregnancy. The content of noradrenaline(NE), expressed as ng/per gram wet weight was 194.79?2.85 in the control group and 78.56?0.48 in the late pregnancy group (P