Genetic Analysis of Prosaposin, the Lysosomal Storage Disorder Gene in Parkinson's Disease.

Recent genetic studies clearly indicate that variants in several lysosomal genes act as risk factors for idiopathic Parkinson's disease (PD). Variants in the co-activator of glucocerebrosidase gene (GBA) and the four active saposins (Sap A-D) which are encoded by the prosaposin gene (PSAP) are of particular interest; however, their genetic roles in PD are unknown. Whole-exome sequencing and Sanger sequencing were used to assess the genetic etiology of 400 autosomal dominant inherited PD (ADPD) and 300 sporadic PD (SPD) patients. Variants from public databases, including Genome Aggregation Database-East Asian (GnomAD_EAS) and Chinese Millionome Database (CMDB), were used as control groups. Burden analysis based on gene and domains level were performed to investigate the role of rare PSAP variants in PD. Six rare and likely pathogenic variants, located in the Sap A-D domains, were identified and accounted for 0.75% (3/400) of ADPD and 1.33% (4/300) of SPD in the Chinese population. Based on the gene or domain, burden analysis showed that damaging missense variants in SapC had statistical significance on the risk of developing PD. Interestingly, rs4747203, an intronic variant potentially linked to PSAP expression, was associated with reduced risk for PD (p = 8.6e-7 in GnomAD EAS and p = 0.002 in Chinese). Clinically, patients carrying the likely pathogenic variants presented typical PD motor symptoms and responded well to levodopa treatment. Six out of seven patients carrying the likely pathogenic variants of PSAP presented slow disease progression, and none of the patients developed cognitive impairment. Our study expands the spectrum of mutations associated with the risk of developing PD and enhances the understanding of the relationship of the clinical phenotype of PD with PSAP variants.

miR-146a-5p improves the decidual cytokine microenvironment by regulating the toll-like receptor signaling pathway in unexplained spontaneous abortion.

Spontaneous abortion (SA) is a common pregnancy failure, but the cause of numerous cases remains unexplained. Decidual immune cells (DICs)-mediated cytokine microenvironment is involved in pregnancy and regulated by many microRNAs, but whether microRNA-146a-5p (miR-146a) regulate the decidual cytokine microenvironment and the potential mechanisms in unexplained SA pathogenesis have rarely been reported. In this study, the levels of cytokines and miR-146a in healthy and unexplained SA deciduae were first investigated, and the correlation between them was analyzed. Then, the effect of miR-146a inhibitor on cytokines was assessed in healthy deciduae-derived DICs. Third, the downstream targets and related molecular mechanisms of miR-146a were analyzed by bioinformatics, and the levels of the predicted targets in deciduae were assessed, followed by the correlation analysis between the levels of miR-146a and the targets. Finally, the effect of miR-146a on the predicted targets and inflammatory cytokines was validated in unexplained SA deciduae-derived DICs. As a result, decreased miR-146a correlated with the cytokine disorder in unexplained SA deciduae, and inhibition of miR-146a promoted pro-inflammatory response in healthy deciduae-derived DICs. One hundred four target genes and related molecular mechanisms of miR-146a were predicted, among which the toll-like receptor (TLR) pathway might be associated with the decidual cytokine regulation. Upregulation of miR-146a inhibited the expression of the predicted molecules enriched in the TLR pathway and improved the cytokine disorder in unexplained SA deciduae-derived DICs. Collectively, miR-146a improves the decidual cytokine microenvironment by regulating the TLR pathway in unexplained SA, providing novel potential targets for further therapeutic research.

miR-140 Attenuates the Progression of Early-Stage Osteoarthritis by Retarding Chondrocyte Senescence.

Osteoarthritis (OA) is a major cause of joint pain and disability, and chondrocyte senescence is a key pathological process in OA and may be a target of new therapeutics. MicroRNA-140 (miR-140) plays a protective role in OA, but little is known about its epigenetic effect on chondrocyte senescence. In this study, we first validated the features of chondrocyte senescence characterized by increased cell cycle arrest in the G0/G1 phase and the expression of senescence-associated ß-galactosidase (SA-ßGal), p16INK4a, p21, p53, and γH2AX in human knee OA. Then, we revealed in interleukin 1ß (IL-1ß)-induced OA chondrocytes in vitro that pretransfection with miR-140 effectively inhibited the expression of SA-ßGal, p16INK4a, p21, p53, and γH2AX. Furthermore, in vivo results from trauma-induced early-stage OA rats showed that intra-articularly injected miR-140 could rapidly reach the chondrocyte cytoplasm and induce molecular changes similar to the in vitro results, resulting in a noticeable alleviation of OA progression. Finally, bioinformatics analysis predicted the potential targets of miR-140 and a mechanistic network by which miR-140 regulates chondrocyte senescence. Collectively, miR-140 can effectively attenuate the progression of early-stage OA by retarding chondrocyte senescence, contributing new evidence of the involvement of miR-mediated epigenetic regulation of chondrocyte senescence in OA pathogenesis.

The effect of primary total knee arthroplasty on the incidence of falls and balance-related functions in patients with osteoarthritis.

Knee osteoarthritis (OA) is an established risk factor for falls and balance impairment. This study investigated the incidence of falls, balance-related outcomes and risk factors for falls before and after primary total knee arthroplasty (TKA). Three hundred seventy-six OA patients scheduled to undergo TKA were included. Falls data within the preoperative, first postoperative and second postoperative years were collected, balance-related functions were assessed using the Assessment of Quality of Life (AQoL), WOMAC, Falls Efficacy Scale International (FES-I), Activities-specific Balance Confidence (ABC), knee extension strength, Berg Balance Scale (BBS) and Timed Up and Go (TUG) before surgery and 1 and 2 years after surgery. Compared with preoperative values, the incidence of falls significantly decreased (14.89%, 6.23% and 3.14% within the preoperative, first postoperative and second postoperative years, respectively) and the AQoL, WOMAC, FES-I, ABC, knee extension strength, BBS and TUG significantly improved after TKA. Logistic regression analysis revealed that Kellgren-Lawrence grade ≥ 3 of the contralateral knee was an independent risk factor for falls before and after TKA. Conclusively, primary TKA is associated with a reduced incidence of falls and improved balance-related functions, and the contralateral knee should be considered in the design of fall-prevention strategies in patients with OA.

Correlations between inflammatory cytokines, muscle damage markers and acute postoperative pain following primary total knee arthroplasty.

BACKGROUND: Despite the success of total knee arthroplasty (TKA) in reducing knee pain and improving functional disability, the management of acute postoperative pain is still unsatisfactory. This study was aimed to quantitatively analyze the possible correlations between inflammatory cytokines, muscle damage markers and acute postoperative pain following primary TKA. METHODS: Patients scheduled for unilateral primary TKA were consecutively included, the serial changes of the numerical rating scale (NRS) at rest (NRSR) and at walking (NRSW), serum inflammatory cytokines and muscle damage markers were assessed before surgery (T0) and at postoperative day 1, 2, 3 and 5 (T1-T4, respectively); while pain disability questionnaire (PDQ) and synovial fluid inflammatory cytokines were evaluated at T0. The correlations between inflammatory cytokines, muscle damage markers and pain scores were examined, and Bonferroni correction was applied for multiple comparisons. RESULTS: Ninety six patients were included for serum markers and pain evaluations at T0-T4, while 54 (56.25%) for synovial fluid cytokines at T0. The NRSR at T1 and T2 were positively correlated with preoperative NRSW, while the NRSW at T1 to T4 were positively correlated with preoperative NRSR, NRSW and PDQ (all p < 0.05). The NRSR was positively correlated with serum PGE2, IL-6, and CK at T1; the NRSW was positively correlated with serum CRP at T1, with PGE2 and IL-6 at T1 to T3, with CK at T2 and T4, and with Mb and LDH at T1 to T4 (all p < 0.003). Meanwhile, positive correlations were observed between preoperative NRSW and synovial fluid PGE2, IL-6, IL-8, or TNF-α, as well as between PDQ and PGE2 (all p < 0.003), but no associations between postoperative pain scores and preoperative synovial fluid cytokines was found (all p ≥ 0.003). Additionally, the NRSR at T1 and T2, and NRSW at T1 to T4 were positively correlated with body mass index (all p < 0.05). CONCLUSIONS: Serum inflammatory cytokines and muscle damage markers are positively correlated with acute postoperative pain following primary TKA, and the key cytokines (CRP, PGE2, and IL-6) and markers (Mb, CK and LDH) may serve as the targets for developing novel analgesic strategies.

Control-released basic fibroblast growth factor-loaded poly-lactic-co-glycolic acid microspheres promote sciatic nerve regeneration in rats.

Although peripheral nerve injury may result in a loss of function in innervated areas, the most effective method for nerve regeneration remains to be determined. The aim of the present study was to investigate the effect of control-released basic fibroblast growth factor (bFGF)-loaded poly-lactic-co-glycolic acid (PLGA) microspheres on sciatic nerve regeneration following injury in rats. bFGF-PLGA microspheres were prepared and their characteristics were evaluated. The sciatic nerve was segmentally resected to create a 10 mm defect in 36 Sprague Dawley (SD) rats and, following the anastomosis of the nerve ends with a silicone tube, bFGF-PLGA microspheres, free bFGF or PBS were injected into the tube (n=12 in each group). The outcome of nerve regeneration was evaluated using the sciatic function index (SFI), electrophysiological test and histological staining at 6 weeks and 12 weeks post-surgery. The bFGF-PLGA microspheres were successfully synthesized with an encapsulation efficiency of 66.43%. The recovery of SFI and electrophysiological values were significantly greater (P<0.05), and morphological and histological observations were significantly greater (P<0.05) in bFGF-PLGA microspheres and bFGF groups compared with those in the PBS group, and the quickest recovery was observed in the bFGF-PLGA microspheres group. In conclusion, the bFGF-PLGA microspheres may promote nerve regeneration and functional recovery in the sciatic nerve, and may have potential therapeutic applications in peripheral nerve regeneration.

Exendin-4 Ameliorates Lipotoxicity-induced Glomerular Endothelial Cell Injury by Improving ABC Transporter A1-mediated Cholesterol Efflux in Diabetic apoE Knockout Mice.

ATP-binding cassette transporter A1 (ABCA1), which promotes cholesterol efflux from cells and inhibits inflammatory responses, is highly expressed in the kidney. Research has shown that exendin-4, a glucagon-like peptide-1 receptor (GLP-1R) agonist, promotes ABCA1 expression in multiple tissues and organs; however, the mechanisms underlying exendin-4 induction of ABCA1 expression in glomerular endothelial cells are not fully understood. In this study we investigated the effect of exendin-4 on ABCA1 in glomerular endothelial cells of diabetic kidney disease (DKD) and the possible mechanism. We observed a marked increase in glomerular lipid deposits in tissues of patients with DKD and diabetic apolipoprotein E knock-out (apoE-/-) mice by Oil Red O staining and biochemical analysis of cholesterol. We found significantly decreased ABCA1 expression in glomerular endothelial cells of diabetic apoE-/- mice and increased renal lipid, cholesterol, and inflammatory cytokine levels. Exendin-4 decreased renal cholesterol accumulation and inflammation and increased cholesterol efflux by up-regulating ABCA1. In human glomerular endothelial cells, GLP-1R-mediated signaling pathways (e.g. Ca2+/calmodulin-dependent protein kinase, cAMP/PKA, PI3K/AKT, and ERK1/2) were involved in cholesterol efflux and inflammatory responses by regulating ABCA1 expression. We propose that exendin-4 increases ABCA1 expression in glomerular endothelial cells, which plays an important role in alleviating renal lipid accumulation, inflammation, and proteinuria in mice with type 2 diabetes.

[Endothelium Aging and Oxidative Stress].

In the endothelium, ROS mainly derive from mitochondria, endothelial nitric oxide synthases and NADPH oxidases 4. Excessive ROS are a major cause of oxidative stress, the primary stimulus of vascular dysfunction and oxidative stress-related diseases. However, cellular evolution has made possible the development of adaptive antioxidant systems that scavenge excessive ROS, such as Nrf2/Keapl-ARE, PPAR-y, SIRT and FOXO, etc. Among them, the Nrf2/Keapl-ARE signaling pathway is perhaps the most prominent. What is more, there are the "crosstalk" among these antioxidant stress-related signaling pathways aim to alleviate oxidative stress injurys and promote cells survival. The understanding of the relationship between endothelial aging and oxidative stress may serve as a therapeutic clues in the treatment of oxidative stress-related diseases.

[In vitro analysis and in vivo tracing of BrdU-labeled rat bone marrow mesenchymal stem cells].

OBJECTIVES: To investigate the optimal condition of bromodeoxyuridine (BrdU) labeling for bone marrow mesenchymal stem cells (BMSCs) in vitro and the feasibility of in vivo tracing of BrdU-labeling BMSCs. METHODS: BMSCs were isolated from Wistar rats and were in vitro routinely cultured. The third passage BMSCs was used for identification of special surface antigens by immunohistochemical methods. The purified BMSCs were incubated with BrdU at different concentrations for different incubating time to investigate optimal BrdU concentration and incubating time for cell labeling. The cell labeling index of BrdU was calculated with immunohistochemical analysis. BMSCs labeled BrdU were injected into damaged gastric mucosa of rats by micro injector. The colonization of BMSCs labeled BrdU in gastric mucosa was viewed. RESULTS: After purification and proliferation, the primary cultured BMSCs were uniformly long spindle-shapped form and formed cell colony, which showed the characteristics of stem cell. Immunocytochemistry showed BMSCs were positive for CD44 and CD90, while negative for CD14, CD45. The labeling rate of BrdU increased with the labeling time lasting and reached its height at 48 h. After incubating 48 and 72 hours, the labeling rate of BrdU with a concentration of 10 micromol/L was higher than that of BrdU with a concentration of 5 micromol/L (P < 0.05) and similar with that of BrdU with a concentration of 15 micromol/L (P > 0.05). In addition, the BrdU labeling could be detected after five consecutive passages and the labeling time could keep 21 d. The pathological observation demonstrated that BrdU-labeled BMSCs could colonize the damaged gastric mucosa with normal morphologic characteristics during observation period. CONCLUSION: BrdU labeling might be a feasible method for dynamic observation of the migration, growth and differentiation of migrating BMSCs in colonizing sites.

Pancreas anatomy and surgical procedure for pancreatectomy in rhesus monkeys.

BACKGROUND: The aim of this study was to investigate the pancreas anatomy and surgical procedure for harvesting pancreas for islet isolation while performing pancreatectomy to induce diabetes in rhesus monkeys. METHODS: The necropsy was performed in three cadaveric monkeys. Two monkeys underwent the total pancreatectomy and four underwent partial pancreatectomy (70-75%). RESULTS: The greater omentum without ligament to transverse colon, the cystic artery arising from the proper hepatic artery and the branches supplying the paries posterior gastricus from the splenic artery were observed. For pancreatectomy, resected pancreas can be used for islet isolation. Diabetes was not induced in the monkeys undergoing partial pancreatectomy (70-75%). CONCLUSIONS: Pancreas anatomy in rhesus monkeys is not the same as in human. Diabetes can be induced in rhesus monkeys by total but not partial pancreatectomy (70-75%). Resected pancreas can be used for islet isolation while performing pancreatectomy to induce diabetes.

Monitoring intestinal microbiota profile: a promising method for the ultraearly detection of colorectal cancer.

Colorectal cancer is one of the most common cancers and is very hard to be detected at an ultraearly stage because of lack of valuable predicating methods that often lead to treatment failure. Intestinal microbiota has long been considered to implicate in colorectal cancer pathology; and many recent reports point out a close linkage between the intestinal bacteria and the genesis of the tumor. Present studies indicate that the structure and characteristics of the intestinal microbiota are significantly altered in colorectal cancer, precancerous lesion, and high risk population compared with healthy controls and low risk population. Based on the current studies and theories, we postulate monitoring the intestinal bacterial profile by the molecular methods that could fulfill the ultraearly prediction about the degree of the risk developing into colorectal cancer. Further population-based epidemiological study is useful to reveal the characteristics of the intestinal microbiota in ultraearly colorectal cancer, which might provide some novel prophylactic and therapeutic strategies for the colorectal cancer.

Enhancement of cytotoxicity of antimicrobial peptide magainin II in tumor cells by bombesin-targeted delivery.

AIM: To investigate whether the conjugation of magainin II (MG2), an antimicrobial peptides (AMPs), to the tumor-homing peptide bombesin could enhance its cytotoxicity in tumor cells. METHODS: A magainin II-bombesin conjugate (MG2B) was constructed by attaching magainin II (MG2) to bombesin at its N-terminus. The peptides were synthesized using Fmoc-chemistry. The in vitro cytotoxicity of the peptide in cancer cells was quantitatively determined using the CCK-8 cell counting kit. Moreover, the in vivo antitumor effect of the peptide was determined in tumor xenograft models. RESULTS: The IC(50) of MG2B for cancer cells (10-15 µmol/L) was at least 10 times lower than the IC(50) of unconjugated MG2 (125 µmol/L). Moreover, the binding affinity of MG2B for cancer cells was higher than that of unconjugated MG2. In contrast, conjugation to a bombesin analog lacking the receptor-binding domain failed to increase the cytotoxicity of MG2, suggesting that bombesin conjugation enhances the cytotoxicity of MG2 in cancer cells through improved binding. Indeed, MG2B selectively induced cell death in cancer cells in vitro with the IC(50) ranging from 10 to 15 µmol/L, which was about 6-10 times lower than the IC(50) for normal cells. MG2B (20 mg/kg per day, intratumorally injected for 5 d) also exhibited antitumor effects in mice bearing MCF-7 tumor grafts. The mean weights of tumor grafts in MG2B- and PBS-treated mice were 0.21±0.05 g and 0.59±0.12 g, respectively. CONCLUSION: The results suggest that conjugation of AMPs to bombesin might be an alternative approach for targeted cancer therapy.

Diffusion tensor MRI for the assessment of cerebral ischemia/reperfusion injury in the penumbra of non-human primate stroke model.

OBJECTIVES: The purpose of this study is to use diffusion tensor imaging (DTI) parameters to evaluate cerebral ischemia/reperfusion injury in the infarct core (IC) and ischemic penumbra (IP) in a rhesus transient middle cerebral artery occlusion (MCAO) model. METHODS: Seven rhesus monkeys were used to construct the MCAO model. The temporal evolution of the relative apparent diffusion coefficient (rADC) and the relative fractional anisotropy (rFA) in the IC area, infarct growth area (IG), and reversible penumbra area (RP) were investigated. RESULTS: The rADC increased in the three areas in the early stage of reperfusion (1 hour after the reperfusion). However, the rate of rADC improvement was significantly slower in IG than in IC and RP. Different temporal evolutions of rFA were observed in the three areas in the following stage of reperfusion (3-24 hours after the reperfusion), which continued to decline in IG but slightly elevated in IC and RP. DISCUSSION: These findings suggest that the evolution of DTI parameters can help in the assessment of cerebral ischemia/reperfusion injury in the penumbra and predict the growth of the infarction area after stroke.

Use of pre-, pro- and synbiotics in patients with acute pancreatitis: a meta-analysis.

AIM: To assess the clinical outcomes of pre-, pro- and synbiotics therapy in patients with acute pancreatitis. METHODS: The databases including Medline, Embase, the Cochrane Library, Web of Science and Chinese Biomedicine Database were searched for all relevant randomized controlled trials that studied the effects of pre-, pro- or synbiotics in patients with acute pancreatitis. Main outcome measures were postoperative infections, pancreatic infections, multiple organ failure (MOF), systemic inflammatory response syndrome (SIRS), length of hospital stay, antibiotic therapy and mortality. RESULTS: Seven randomized studies with 559 acute pancreatic patients were included. Pre-, pro- or synbiotics treatment showed no influence on the incidence of postoperative infections [odds ratios (OR) 0.30, 95% confidence interval (CI): 0.09-1.02, P = 0.05], pancreatic infection (OR 0.50, 95% CI: 0.12-2.17, P = 0.36), MOF (OR 0.88, 95% CI: 0.35-2.21, P = 0.79) and SIRS (OR 0.78, 95% CI: 0.20-2.98, P = 0.71). There were also no significant differences in the length of antibiotic therapy (OR 0.75, 95% CI: 0.50 - 1.14, P = 0.18) and the mortality (OR 0.75, 95% CI: 0.25-2.24, P = 0.61). However, Pre-, pro- or synbiotics treatment was associated with a reduced length of hospital stay (OR -3.87, 95% CI: -6.20 to -1.54, P = 0.001). When stratifying for the severity of acute pancreatitis, the main results were similar. CONCLUSION: Pre-, pro- or synbiotics treatment shows no significant influence on patients with acute pancreatitis. There is a lack of evidence to support the use of probiotics/synbiotics in this area.

[Toxic effect of immunosuppression agent Everolimus on insuloma cells and pancreatic islets of SD rat].

OBJECTIVE: To investigate the effect of immunosuppression agent Everolimus on the viability and function of insuloma cells (INS-1) and pancreatic islet cells cultured in vitro. METHODS: INS-1 cells and islets were treated with a series of concentrations of immunosuppression agents (Everolimus, Cyclosporin A, Sirolimus and Mycophenolate Mofetil). The viability of INS-1 cells and rat pancreatic islets were determined with MTT and the function of INS-1 cells and rat pancreatic islets was evaluated with Glucose-stimulated insulin secretion assay. RESULTS: Above the clinical blood concentration, the inhibition rate of islet cell proliferation in the high concentration group of Everolimus and Sirolimus was significantly lower than that of Cyclosporin A and Mycophenolate Mofetil group (P < 0.05); Everolimus in the blood drug level, like other immunosuppressive agents, can inhibit the function of insulin secretion, and the stimulation index of each group was no significant difference. CONCLUSION: Compared to Mycophenolate Mofetil and Cyclosporin A, Everilimus and Sirolimus demonstrate lower toxicity effect on INS-1 cells and rat pancreatic islets in vitro and Everolimus is expected as a new type of immunosuppressive agent used in clinical islet transplantation.

Practical value of applying cdc2 kinase shRNA to chronic proliferative cholangitis in treatment of hepatolithiasis.

BACKGROUND/AIMS: High stone recurrence and biliary restenosis rates in hepatolithiasis patients have been confirmed to be closely related to postoperatively-remnant chronic proliferative cholangitis (CPC), but effective management strategies have not yet been developed. Since CPC is a type of hyperplastic disease, this study was designed to investigate inhibitory effectiveness of cdc2 k ShRNA on hyperplastic behavior and lithogenic potentiality of CPC. METHODOLOGY: 0.5 ml of P-cdc2 shRNA was injected transpapillarily into the bile duct lumen in a rat model of cholangitis. Then, the effects of cdc2 k ShRNA on CPC were evaluated by histology, immunohistochemistry, RT-PCR, Western blot, biochemistry and enzymatic histochemistry for cdc2 k, PCNA, Ki-67, Procollagen III, Mucin 5AC, beta-glucuronidase and hydroxyproline. RESULTS: cdc2 k shRNA-3 treatment could efficiently inhibit hyperplasia of biliary epithelium, submucosal gland, and collagen fiber by inhibiting mRNA and protein expressions of the proliferation-related gene, cdc2 k, PCNA and Ki-67, thus holding the promise to control or reverse CPC and its secondary biliary stricture. Also of note, this novel treatment may decrease the lithogenic potential of CPC via inhibition of endogenous beta-glucuronidase and Mucin 5AC expression, hereby facilitating the prevention of stone recurrence. CONCLUSION: cdc2 k shRNA-3 treatment could effectively inhibit the hyperplastic behavior and lithogenic potentiality of CPC, which might help to prevent the biliary restenosis and stone recurrence.

[Effects of enteral nutrition and parenteral nutrition on gut epithelial tight junction and barrier function in rats after surgical stress].

OBJECTIVE: To compare the effects of enteral nutrition (EN) and parenteral nutrition (PN) on gut mucosal barrier function and intestinal epithelial tight junctions in rats after surgical stress. METHODS: Fifty SD rats with surgical trauma were randomly divided into three groups: total parenteral nutrition (TPN) group and enteral nutrition (EN) group with isocaloric and isonitrogenous nutrition and placebo group. Nutrients were administered via the neck vein and needle jejunostomy for the TPN group. The homogenated tissues of liver, lung, and mesenteric lymph nodes were cultured to determine the bacterial translocations rates. The transmembrane binding proteins (occludin) were measured with immunohistochemistry. The ultrastructure and morphology of intestinal epithelial tight junctions were observed by electron microscope. The feces in cecum were cultured for anaerobic bacterial growth analyses. RESULTS: The EN group had more lactobacteria and bifydobacteria than the TPN group, but not statistical significant. The EN group had greater expression of occludin in the intestines than the TPN group. Furthermore, the intestinal epithelial tight junction and microvilli of the EN group were more intact compared with those of the TPN group. The bacterial translocations rates of liver, lung and mesenteric lymph nodes were significantly lower in the EN group than in the TPN group. CONCLUSION: Neither EN nor standard PN maintains intestinal membrane barriers. But EN increases the expression of transmember binding proteins, maintains the gut epithelial tight junction, improves intestinal mucosal barrier and reduces gut bacterial translocation.

[Effects of the different culture and isolation methods on the growth, proliferation and biology characteristics of rat bone marrow mesenchymal stem cells].

OBJECTIVE: To explore a suitable method for isolation, purification and multiplication of bone marrow mesenchymal stem cells (BMSCs). METHODS: Density gradient centrifugation and adherence separation methods were applied for isolation of BMSCs from Wistar rats. The cells were cultured and proliferated in culture medium containing calf serum (CS), fetal bovine serum (FBS), free of serum or different volume fraction of FBS. The characteristic and the morphology of BMSCs were observed under inverted microscope every day. The growth curves were draw and the surface antigen of BMSCs were detected by immunocytochemistry technique. The microstructure was observed by transmission electron microscope (TEM). RESULTS: The pure primary cells can be procured by density gradient centrifugation. But the primary cells cultured by adherence separation methods demonstrated higher cytoactive, more rapid proliferation, earlier colony confluence and shorter time for passage than that cultured by density gradient centrifugation method. The cells by adherence separation methods were essentially purified at passage 4. Both CS and FBS can promote the growth and proliferation of BMSCs, but the colony forming efficacy of cells (46.50%) cultured in medium containing 0.12 volume fraction FBS was the highest. The cells surface markers CD44, CD90 were positive and CD14, CD45 were negative. BMSCs were observed by TEM and possessed the characteristic of stem cells. Conclusion BMSCs with high quality and activity can be obtained with adherence separation by suitable method and culture conditions. L-DMEM medium containing 0.12 volume fraction FBS showed more profitable for the growth and proliferation of BMSCs.

Significance of controlling chronic proliferative cholangitis in the treatment of hepatolithiasis.

BACKGROUND: Recently, high stone recurrence and biliary restenosis rates in hepatolithiasis patients have been confirmed to be closely related to chronic proliferative cholangitis (CPC). However, the effective management of CPC has not yet been established. METHODS AND RESULTS: A vicious cycle exists between the presence of intrahepatic calculi and CPC: both the stone itself and secondary biliary infection can stimulate persistent hyperplasia in the biliary duct wall, leading to the occurrence of CPC and biliary stricture. The recurrent attacks of CPC will, in turn, facilitate new stone formation via mucoglycoprotein production, or induced biliary stricture and cholestasis. Thus, even when the stone is completely removed and the biliary tract stenosis is corrected, residual CPC will persist and progress, with an underlying risk for postoperative stone recurrence and biliary tract restenosis. Therefore, the perfect hepatolithiasis treatment would target stone removal and correction of the biliary tract stricture, as well as control of postoperative residual CPC. In fact, CPC, the management of which has been traditionally ignored, is the key to breaking this vicious cycle. CONCLUSIONS: Overall, the subsequent treatment of residual CPC after operation or choledochoscopic lithotomy would be helpful to decrease postoperative stone recurrence and the rate of biliary restenosis. Adding such treatment would reduce the incidence of surgical reintervention and choledochoscopic lithotomy, and it would also improve the postoperative hepatolithiasis outlook.