Object To study the effect of cryopreservation on the ability of hematopoietic stem cells from human bone marrow and the action of total saponins of Panax ginseng (TSPG) to reduce their freezing injury. Methods Gradual cooling methods were used to place mononuclear cells (MNC) in liquid nitrogen with 5% dimethylsulfoxide (DMSO) for 3—5 months. After thawed, the biological properties of MNC were monitored, which included the mean trypan blue exclusion rate and the mean recovery rate of MNC, CFU-Mix, CD34+ cell, and total cells; Thawed MNC were cultured with various concentrations of TSPG, the effect of TSPG on the recoverable ability from cryopreservation damage were detected by colony-forming assay, colorimetric MTT assay for cell proliferation and flow cytometry. Results A fraction of MNC lost their proliferatory ability after thawed, but the damage was not deteriorated with freezing time. TSPG (25 ?g/mL) could raise the colony production rate of thawed hematopoietic stem cells; TSPG (25—50 ?g/mL) could raise the colony production rate of thawed hematopoietic stem cells; TSPG (25—50 ?g/mL) could improve their proliferation; TSPG (25 ?g/mL) could also promote the entray of them into cell proliferatory cycle. Conclusion TSPG could induce the proliferation of thawed hemato- poietic stem cells and raise the post-freezing recoverable ability of hematopoietic stem cells.

Objective: To observe the preventive effect of alkaline drinking water on hyperuricemia in mice. Methods: Sixty male SPF Kunming mice were randomly divided into six groups: pH 7.3, pH 8.0, pH 9.3 intervention groups, in which the mice were given water with pH values of 7.3±0.5, 8.0±0.5 and 9.3±0.6, respectively; the control group, model group and positive drug group ( with 2 g/L allopurinol ) were given double distilled water. Except for the control group, the mice in each group were given yeast by gavage (1.5 g/mL) for 13 days. On the 14th day, the mice were injected with 300 mg/kg potassium oxyzinate by intraperitoneal injection, and then fasted for 1 day. On the 16th day, serum uric acid, creatinine and urea nitrogen were detected, and renal tissues were stained to observe the morphology.The expression levels of neutrophil gelatinase-associated lipocalin ( NGAL ), tissue inhibitor of metalloproteinase 1( TIMP1 ), organic anion transporter 1 ( OAT1 ) and urate transporter 1 ( URAL-1 ) in renal tissues were determined bywestern blotting. The mRNA expression levels of URAL-1 and OAT1 were detected by real-time fluorescent quantita⁃tive polymerase chain reaction. Results: The level of serum uric acid was higher in the model group than in the control group and in the pH 9.3 intervention group (both P<0.05). The number and area of renal tubular lesions were less in the pH 9.3 intervention group than in the model group (all P<0.05). The relative expression levels of NGAL and URAT-1 proteins were lower in the pH 9.3 intervention group than in the model group, and the relative expression level of OAT1 protein was higher in the pH 9.3 intervention group than in the model group ( all P<0.05). The relativeexpression level of URAT-1 mRNA was lower in the pH 9.3 intervention group than in the model group, and the rela⁃tive expression level of OAT1 mRNA was higher in the pH 9.3 intervention group than in the model group ( all P<0.05 ). Conclusion Alkaline drinking water with pH value of 9.3±0.6 can effectively prevent hyperuricemia and acute kidney injury in mice.

Objective To investigate the intestinal pathophysiological mechanism of bacterial translocation and endotoxemia in rabbits with acute spinal cord injury (SCI). Methods Paraplegia was induced by injuring the spinal cord of 30 rabbits by the method of Fehlings. Twelve rabbits were used for recording the changes of gastrointestinal (GI) electrophysiology and colon pressure. The left 18 rabbits were experimental group and were killed in 24, 48 and 72 h after injury. The other 6 rabbits served as normal group. Under aseptic condition, samples of blood and mesenteric lymph node were collected for bacterial cultures and endotoxin detection. The small intestines were observed by light and electron microscopy. The colons were inspected by light microscopy. Results After SCI, the electrophysiology of the GI tract was changed especially at the middle and distal colon. The peristalsis of the middle and distal colon was reduced and sometimes even disappeared. In the early stage, the main pathology was hyperemia of blood vessel and infiltration of inflammatory cells. The interepithelial tight junctions became wider and the columnar epithelium was disintegrated. All of the pathological changes may lead to the destruction of the intestinal barrier. The endotoxin level were increased since 24 h after SCI and had statistically significant difference compared with that at 72 h (P0.05). Conclusion After SCI, the middle and distal colon dysfunction induces constipation, bacterial overgrowth, and blood flow congestion. These factors may accelerate the destruction of the intestinal barrier and lead to bacterial translocation and endotoxemia.

Objective To investigate the effect of electrical stimulation of S_3 nerve root on improvement of intestinal mucosal barrier function in rabbits with acute complete spinal cord injury. Methods Model of paraplegia was built by injuring spinal cord in rabbits. Then, the rabbits with electrical stimulation of S_3 nerve root were set as experimental group and those without set as control group. Normal rabbits were set as normal group. Under aseptic condition, portal vein blood was collected for quantitative determination of endotoxin and bacterial culture ; and liver, spleen and mesenteric lymph nodes were collected for bacterial culture and strain identification. Liver, spleen, mesenteric lymph nodes and small intestines were collected from experimental group and control group for pathological HE staining; while small intestine were observed by light and electron microscopes. Results In control group, the intestinal mueosal barrier and the other organs were destroyed obviously, with higher level of Serum endotoxin and higher rate intestinal flora translocation than that in experimental group and normal group. In the experimental group, the electrical stimulation of S_3 nerve root could improve motility of the denervated intestine, with more defecation content, less destruction of the intestinal mucosa and lighter other organ damage compared with control group, serum endotoxin level was significantly reduced compared with control group but showed no statistical difference compared with normal group, with obvious decrease of bacterial translocation rate. Conclusions After spinal cord injury in rabbits, electrical stimulation of S_3 nerve root can facilitate intestinal tract motility, improve intestinal mucosal barrier function and hence alleviate endotoxemia and intestinal bacterial translocation, as is beneficial to reducing SIRS and MOBS.

Objective To investigate the expression of tumor suppressor gene p16~(INK4A) in mouse endometrium during the estrus cycle and early pregnancy and its possible role in blastocyst implantation. Methods Reverse transcription polymerase chain reaction(RT-PCR) was used to detect the expression of p16~(INK4A) mRNA,immunohistochemistry and Western blotting were applied to detect p16~(INK4A) protein in mouse endometrium tissues during the estrus cycle and early pregnancy. Results The intensity of p16~(INK4A) mRNA expression in mouse early pregnancy was higher than that in the estrus cycle.Compared with the other 3 stages, the level of p16~(INK4A) mRNA expression at estrus was obviously higher. During the early pregnancy, the level of p16~(INK4A) mRNA expression increased steadily from day 2 to day 5,reaching the maximal level on day 5,then decreasing. Both immunohistochemical and Western blotting analysis showed the same results in expression patterns of p16~(INK4A) protein for mouse endometrium tissues as those results of RT-PCR.Conclusion p16~(INK4A) is involved in the embryos penetrating into the endometrial barrier.

Purpose To study the expression of TMPRSS2, ERG and ETV1 in prostatic cancer and their clinical pathologic signifi-cance. Methods Tissue microarray and immunohistochemistry (MaxVision) were used to detect TMPRSS2, ERG and ETV1 expres-sion in 70 prostatic cancer tissues, 10 prostatic intraepithelial neoplasia tissues and 18 benign prostate tissues. Results There was no statistical significance on positive rate of the expression of TMPRSS2 among prostatic cancer tissues, prostatic intraepithelial neoplasia tissues and benign prostate tissues (P>0. 05). The positive rate (81. 4%) of ERG in prostatic cancer tissues was significantly higher than that in prostatic intraepithelial neoplasia tissues ( 30. 0%) and benign prostate tissues ( 0. 0 ) ( P <0. 05 ) . The positive rate (68. 6%) of ETV1 in prostatic cancer tissues was significantly higher than that in prostatic intraepithelial neoplasia tissues (50. 0%) and benign prostate tissues (22. 2%) (P<0. 05). There was no correlation among the positive rates of TMPRSS2, ERG and ETV1 in prostatic cancer tissues and age (P>0. 05). The expression of TMPRSS2, ERG and ETV1 was positively correlated to Gleason score and clinical stage (P<0. 05). TMPRSS2 had positive correlation with ERG (rs =0. 465, P<0. 01). TMPRSS2 had positive correla-tion with ETV1 (rs =0. 590, P<0. 01). ERG had no positive correlation with ETV1 (rs =0. 151, P>0. 05). Conclusion ERG and ETV1 are expected to become therapeutic targets for prostate cancer. Detecting TMPRSS2, ERG and ETV1 at the same time is helpful to diagnosis and differential diagnosis of prostatic cancer, which might be new molecule markers of prostate cancer.

Objective:To investigate the diagnostic value of different detection methods for Mycobacterium tuberculosis in bronchoalveolar lavage fluid (BALF) from patients with pulmonary tuberculosis.Methods:BALF from l00 patients in Changsha Central Hospital from January 2013 to December 2015 was collected.Among 100 patients,65 cases were clinically diagnosed as tuberculosis,and 35 cases served as control.BALF smear method,polymerase chain reaction (PCR) and membrane reverse dot blot (RDB) were used for synchronous detection of Mycobacterium tuberculosis.Results:The positive rates by BALF smear method,PCR and RDB were 43.08％,73.84％ and 92.31％,respectively (P＜0.05).Sensitivity,specificity,accuracy,and negative predictive value for BALF smear were 43.08％,88.57％,59.00％,and 45.59％,respectively;for PCR were 73.85％,100％,83.00％,and 67.31％,respectively;for RDB were 92.31％,100.00％,95.00％,and 87.50％,respectively.Conclusion:The technique of membrane RDB can not only accurately diagnose Mycobacterium tuberculosis,but also can rapidly and easily identify the resistance of Mycobacterium tuberculosis to streptomycin (SM),rifampicin (RFP) and isoniazid (INH) genotypes.It possesses high clinical value.

OBJECTIVEThe molecular targets of ginsenoside Rg1-induced neural stem cells (NSCs) differentiation were screened by genechip.

METHOD7th day following ginsenoside Rg1 induced human neural stem cells to neurons the gene expression was observed by genechip. The purpose gene and signal transduction pathways were selected by the data calculations, and then confirmed by western blot and immunohistochemical method.

RESULT7th day following Rg1-induced NSCs differentiation, there were about 675 different genes, 255 genes of which were up-regulated and 420 genes down-regulated obviously. Meanwhile the ERK1/2 (extracellular signal-regulated protein kinase) in MAPK (mitogen-activated protein kinase) pathway was related with the NSCs differentiation. The Western blot and immunohistochemistry detection confirmed that ERK 1/2 protein and its phosphorylation were significantly increased, which can be blocked by PD98059 (ERK1/2 inhibitor). In addition, differentiation rate of NSCs was also decreased obviously in ginsenoside Rg1-induced differentiated NSCs when ERK blocker PD98059 was used.

CONCLUSIONERK1/2 is an important molecular target in ginsenoside Rg1-induced NSC differentiation. The selected differentially expressed genes by genechip may provide new clues to study of ginsenoside Rg1-induced NSCs differentiation.

Cell Differentiation ; drug effects ; Cell Line ; Down-Regulation ; drug effects ; Flavonoids ; pharmacology ; Ginsenosides ; pharmacology ; Humans ; MAP Kinase Signaling System ; drug effects ; genetics ; Neural Stem Cells ; cytology ; drug effects ; metabolism ; Oligonucleotide Array Sequence Analysis ; Protein Kinase Inhibitors ; pharmacology ; Time Factors

Objective To identify the candidate genes in the vicinity of a susceptibility locus (urinary albumin 1,UA-1) contributing to the development of albuminuria in type 2 diabetic KK/Ta mice. Methods Total RNA was extracted from the kidneys of KK/Ta (n=3) and BALB/c (n=2) mice at 20 weeks of age.The gene expression profile in kidney was investigated using the Affymetrix Murine Genome U74Av2 array.Competitive RT-PCR was used to confirm the differential expression of syndecan-4 which located in the vicinity of UA-1.Genome DNA was extracted from KK/Ta and BALB/c mice.DNA sequence analysis of the coding and promotor region of syndecan-4 gene was conducted. Results In the vicinity of the susceptibility locus (UA-1)contributing to the development of albuminuria in type 2 diabetic KK/Ta mice,10 candidate genes that showed differential expression were identified.Among them,the gene expression of syndecan-4in KK/Ta kidneys at 20 weeks of age was up-regulated by 26.1 times of age-matched BALB/c kidneys.Sequence analysis revealed two synonymous polymorphisms in the coding region (A93C and T216C) and three polymorphisms in the promoter region (-T263C,-T396C and -G669A) of the syndecan-4 gene.The TATA box was found at 321 bp upstream from the transcription start site,and the T263C polymorphism was located in the binding site of transcription factor Clox.Conclusions Syndecan-4 gene is mapped in the vicinity of the susceptibility locus contributing to the development of albuminuria in type 2 diabetes.The gene expression of syndecan-4 in KK/Ta kidneys is up-regulated than that in age-matched BALB/c kidneys at 20 weeks of age.Thus syndecan-4 may be one of the potential candidate genes responsible for diabetic nephropathy.Sequence differences in the promoter region influence the expression levels of syndecan-4 genes in KK/Ta kidneys.

Montreal cognitive assessment(MoCA, Beijing Version) was chosen as cognition assessment implement. 63 patients suffering from type 2 diabetes mellitus with mild cognitive impairment (MCI) were chosen to form a research group, and 27 patients with type 2 diabetes mellitus and normal cognitive function served as a control group. It was found that atherosclerosis played an important role in the pathogenesis of MCI in type 2diabetes, therefore, early prevention and management of atherosclerosis may help to improve the cognitive function.