Objective In the present study,we use GC/MS-based metabolomics approaches to make analysis of serum metabolic profiles of healthy people and the hyperactivity of liver yang type of constitution in patients with essential hypertension.Try to establish the discriminant model,to discover biomarkers (group) of the hyperactivity of liver yang type of essential hypertension,and to explore the essential material basis of Traditional Chinese medicine syndrome theory.Methods Classified according to the guiding principles of Chinese medicine research,the hyperactivity of liver yang type of constitution in male patients with essential hypertension (n=18),as well as health volunteers (n=15) were randomly selected from Guang An Men Hospital clinic,wards and medical center in the first half of 2010,selected patients with essential hypertension requirements are not taking any drugs or Chinese herbs,or stop taking the various drugs more than one week.Extracted Venous Blood of subjects fasting for 12 hours,and serum was separated through centrifugation.Serum samples are stored and at-86℃ refrigerator.Survey and evaluate endogenous metabolism in serum samples of health control group and types of syndrome mentioned above by gas chromatograph mass spectrometry (GCMS)analysis.Then,analyze the metabolites with Partial Least Squares-Discriminant Analysis.Further use PCA to analyze the principal component factor loadings matrix analysis,and for variable scatter plot (Loading plot),significant increase or decrease the variables can be found from the figure.The combination of these variables is the lesion biomarkers group.Results Compared with the health control group,13 differentially expressed metabolites in the essential hypertension hyperactivity of liver yang type group can be identified (P＜0.05).8 metabolites were up-regulated expression:Uric acid,citrate,Octadecanoic acid,Hexadecanoic acid,Octadecadienoic acid,Leucine,Cholesterol,Norvaline,and 5 metabolites were down-regulated expression:arachidonate,Oleate,Alanine,Aspartic acid,glycine.Conclusion We are incline to regard that the 13 of EH patient serum differentially expressed metabolites are EH hyperactivity of liver yang syndrome metabolic biomarkers:Uric acid,citrate,Octadecanoic acid,Hexadecanoic acid,Octadecadienoic acid,Leucine,Cholesterol,Norvaline,Arachidonate,Oleate,Alanine,Aspartic acid,glycine.

OBJECTIVE: To establish a method to isolate, culture and identify bone marrow-derived mesenchymal stem cells (BM-MSCs) from inbreed line miniature pig of Wuzhishan (ILMW) in vitro, to compare the biological characteristics of BM-MSCs derived from different pigs, and to supply BM-MSCs for investigating the repair mechanisms of renal injury in ILMW aft er unilateral ureteral obstruction. METHODS: Four or 10-months old ILMW (n=4 per group) were selected and 5 mL of bone marrow fluid was extracted at 1 cm position from iliac wing edge. Mononuclear cells were isolated by density gradient method, then were cultured in the complete medium containing 3 different kinds of fetal bovine serum (FBS) (10% FBS, 12% FBS or 15% FBS). The cells of Passage 1, Passage 3, Passage 5 or Passage 11 were collected to examine biological characteristics including morphology, phenotype, differentiation ability, growth curve and cell cycle. RESULTS: The BM-MSCs were attached to the culture dishes, which were fibroblast-like or whirlpoollike. Primary cultured cells began the adherence at 18 h and entered a logarithmic phase in the 6th day. Eighty percent of them were fused in the 9th day. There were no obvious anomalies in the subcultured cells. The expressions in cell surface antigens of CD29, CD44 and CD90 were positive, while the expressions of CD34 and CD45 were negative. There was no statistically significant difference between cells from different generations (all P>0.05). Under condition of osteogenic induction, alizarin red staining was positive at the 18th day, and alcian blue staining was positive at the 21th day. Cell cycle examination showed that the rate of G0/G1 was about 81.45%. CONCLUSION BM-MSCs of ILMW has advantages of earlier adherent time, more active proliferation, shorter cell subculture cycle, and stable biological characteristics after subculture, which is one of the best kinds of BM-MSCs coming from swine in mainland of China.
Animals ; Cell Differentiation ; Cell Division ; Cell Separation ; Cells, Cultured ; China ; Mesenchymal Stem Cells ; cytology ; Swine

Objective To obtain the basic data of Bama minipigs and provide basic reference for embryo-fetal developmental toxicity study. Methods Pregnant minipigs were sacrificed at different days during the gestation period respectively. The examinations included necropsy,count of corporea lutea,live and dead implantations,fetal body weight, and external,visceral, and skeletal examination of fetuses. Results The basic data of Bama minipigs, such as body weight, fetal development, and fetal malformation/variation were obtained. Conclusions We obtained the basic reproductive parameters of pregnant Bama minipigs and the indexes of fetal development, which can provide valuable reference data for embryo-fetal developmental toxicity tests of Bama minipigs.

To establish a method for isolation, culture and identification of adipose-derived mesenchymal stem cells (ASCs) from the inbreed line miniature pig of Wuzhishan (ILMW).
 Methods: A total of 100 g adipose tissues were obtained from subcutaneous tissues of neck in six-month old healthy ILMW (3 samples, male). ASCs from ILMW (ILMW-ASCs) were isolated from adipose tissues through 0.1% collagenase digestion. The cells at the 3rd, 5th, 8th, 13th passages were collected. Cell morphology, size, phenotype, cell cycle, and apoptosis were monitored. Cell differentiation was induced and cell proliferation curve was drawn.
 Results: The ILMW-ASCs, fibroblast-like or whirlpool-like, began the adherence at 36 h and entered a logarithmic phase in the 5th day. Eighty percent of them were fused in the 7th day. The average diameter and volume of ILMW-ASCs were (17.00±0.54) µm and (2.58±0.24)×10-9 L, respectively. The expressions of CD29, CD44 and CD90 were positive, and there was no significant difference between the different passages (all P>0.05). The expressions of CD45, CD8a and HLA-DR were increased with the increase in passages after the 3th passage (all P<0.05). The adipogenic induction of ILMW-ASCs was observed by positive oil red O staining, and the osteogenic induction of ILMW-ASCs was determined by positive alizarin red staining. Apoptosis and senescence occurred in the 13 passage of ILMW-ASCs, and the proportion of S phase of cell cycle was lower than that in lower passages (all P<0.05). 
 Conclusion: ILMW-ASCs are one of the best choice for porcine ASCs, which might provide a source of candidate stem cells for therapy of large animal disease models and tissue or organ repairment.
Adipose Tissue ; Animals ; Cell Differentiation ; Cells, Cultured ; Male ; Mesenchymal Stem Cells ; Swine ; Swine, Miniature

To isolate bone marrow mesenchymal stem cells (BM-MSCs) and establish the model of chronic kidney disease (CKD) of Wuzhishan (WZS) mini-pig, and to study the repairment effect of BM-MSCs on CKD-induced renal fibrosis in vitro.
 Methods: Density gradient method was used to isolate and culture BM-MSCs. The cells were verified by morphology, phenotype, differentiation and so on. The left partial ureteral obstruction (LPUUO) was used to establish the CKD model, which was evaluated by B-ultrasound, single-photon emission computed tomography (SPECT), HE and Masson staining. The cells were divided into 3 groups, the tissue plus BM-MSCs group, the tissue group, and the BM-MSCs group, respectively. Seven days later, the supernatants were collected to observe the changes of hepatocyte growth factor (HGF) cumulative release. HE and Masson staining was used to observe the changes of renal tissue.
 Results: The isolated BM-MSCs possessed the features as follow: fibroblast-like adherent growth; positive in CD29 and CD90 expression while negative in CD45 expression; osteogenic induction and alizarin red staining were positive; alcian blue staining were positive after chondrogenic induction. Twelve weeks after the operation of LPUUO, B-ultrasound showed the thin renal cortical with pelvis effusion; SPETCT showed the left kidney delayed filling and renal impairment. The accumulation of HGF in the tissue plus BM-MSCs group was significantly higher than that in the tissue alone group at the 1st, 5th, 6th, 7th day, respectively (P<0.05). HE staining showed the different degree of renal lesions between the tissue plus BM-MSCs+CKD group and the tissue alone group, which was aggravated with the time going. Masson staining showed that the cumulative optical density of blue-stained collagen fibers in tissue plus BM-MSCs group was significantly lower than that in the tissue group at the 5th to 7th day (P<0.05).
 Conclusion: BM-MSCs from WZS mini-pig can inhibit or delay the progress of CKD-induced renal fibrosis through autocrine HGF in vitro.
Animals ; Autocrine Communication ; physiology ; Bone Marrow Cells ; Cells, Cultured ; Fibrosis ; physiopathology ; prevention & control ; Hepatocyte Growth Factor ; metabolism ; Kidney ; drug effects ; pathology ; physiopathology ; Mesenchymal Stem Cells ; drug effects ; Renal Insufficiency, Chronic ; complications ; physiopathology ; Swine ; Swine, Miniature ; Ureteral Obstruction ; complications