Collagen (Coll), as the basic material of matrix scaffolds for cell growth, has been widely used in the field of tissue engineering and regenerative medicine. In this study, collagen protein was modified by L-lysine (Lys), and cross-linked by genipin (GN) to prepare the L-lysine-modified collagen (Lys-Coll-GN) scaffolds. Microstructure, pore size, porosity, stability and biocompatibility of Lys-Coll-GN scaffolds were observed. The results showed that the bond between L-lysine and collagen protein molecule was formed by generating amide linkage, and mouse embryo fibroblasts proliferation was not inhibited in the Lys-Coll-GN scaffolds. In the multiple comparisons of Coll-scaf- folds, Coll-GN scaffolds and Lys-Coll-GN scaffolds, Coll-scaffolds was the worst in mechanical characteristics while the highest in biodegradation rate. Compared to Coll-GN scaffolds, Lys-Coll-GN scaffolds had more fiber structure, higher interval porosity (P<0. 01). Although the tensile stress of Lys-Coll-GN scaffolds reduced significantly, its e- longation length extended when the scaffolds was fractured (P<0. 01). The percentage of Lys-Coll-GN scaffolds residual weight was lower than that of Coll-GN scaffolds after all the scaffolds were treated by collagenase for 5 days (P<0. 01). This study suggested that Lys-Coll-GN scaffold had good biocompatibility, and it improved the mechanical property and degradation velocity for collagen-based scaffold. This study gave a new predominant type of tissue engineering scaffold for the regenerative medicine.
Animals ; Biocompatible Materials ; chemistry ; Cell Proliferation ; Collagen ; chemistry ; Cross-Linking Reagents ; Fibroblasts ; cytology ; Iridoids ; chemistry ; Lysine ; chemistry ; Mice ; Porosity ; Tissue Engineering ; Tissue Scaffolds

Objective To observe the proliferation and phenotype-switching of pulmonary arterial smooth muscle cell (PASMC) induced by hypoxia and interfered by Ad-PKGIα. And to investigate the potential regulative role of PKGIα gene in the molecule mechanism of hypoxia pulmonary vessel remodeling (HPVR). Methods To establish the pure PASMC cultured by tissue-sticking methods. Semi-quantitative reverse transcription and polymerase chain reaction (RT-PCR) and Western blot were used to examine the PKGIα mRNA and protein expression after PASMC were transfected by Ad-PKG. The mRNA and protein expressive change of smooth muscle α actin(SM-α-actin) determined the degree of cell phenotype-switching. The changes of PASMC proliferation were determined by flow cytometry and ~3H-TdR incorporated way. Results Ad-PKGIα could transfect into PASMC and highly express. Hypoxia down-regulated the expression of SM-α-actin protein (44. 25±5.34 in normoxia, 32. 18±4. 19 in 12 h hypoxia condition, 21.90 ±2. 44 in 24 h hypoxia condition, P < 0. 05), that could be blocked by the transfeetion of Ad-PKGIα. Hypoxia could push PASMC mitosis and proliferating(~3H-TdR incorporated way: 7570 ± 371 in normoxia,12 020± 831 in 12 h hypoxia condition,14 924 ± 1491 in 24 h hypoxia condition, P <0. 05), that could be blocked by the transfection of Ad-PKGIα, too. Conclusions The results suggested that PKGIα signaling pathway might play an important role in the molecule mechanism of HPVR. And PKGIα gene might be a target point of gene therapy.

AIM:To investigate the role of bcl-2 and bax expression in apoptosis of pulmonary artery smooth muscle cells(PASMC) of rats induced by Na +/H + exchanger isoform-1(NHE-1) inhibition in vitro. METHODS:Intracellular Ca 2+ concentration ([Ca 2+ ]i) was measured using Fura-2/AM. The expression of bcl-2 and bax mRNA in cells were detected by sqRT-PCR, and the expression of Bcl-2 and Bax protein were examined immunohistochemically. RESULTS:The expression of bax mRNA and Bax protein and [Ca 2+ ]i in cells transfected with NHE-1 ribozyme gene increased significantly compared with those of cells transfected with pLXSN and nontransfected control. Meanwhile, the expression of bcl-2 mRNA and Bcl-2 protein in cells transfected with NHE-1 ribozyme gene decreased significantly. CONCLUSION:Apoptosis of pulmonary artery smooth muscle cells induced by inhibiting NHE-1 may have relevance to the increase in [Ca 2+ ]i and bax expression and the decrease in bcl-2 expression.

Objective To explore the correlation between axon guidance factor Semaphorin 5A and clinicopathological features and its role in the invasion and metastasis of gastric cancer.Methods The expression of Semaphorin 5A in gastric cancer tissues of 171 patients with different gender,age,histological type and TNM stage was detected with immunohistochemistry assay.The expression of Semaphorin 5A was determined by Western blotting assay in gastric cancer cell lines SGC7901 and MKN-45 with metastatic ability and gastric cancer cell lines SNU-1 and AGS without metastatic ability.With RNA interfere technique(RNAi),Semaphorin 5A siRNA expression vector was constructed and transfected into gastric cancer cell line SGC7901.The stable gastric cancer cell line down-expressing Semaphorin 5A was established.The effect of Semaphorin 5A gene silencing on the adhesion,migration and invasion of gastric cancer cell was examined by cell adhesion test,wound healing test and transwell chamber assays.Results The expression level of Semaphorin 5A was correlated with the differentiation degree of gastric cancer(x2 =6.32,P =0.01),lymphnode metastasis(x2 =7.68,P=0.01)and distant metastasis of gastric cancer(x2 =13.67,P =0.00),not correlated with age(x2 =0.21,P=0.79),gender(x2=1.79,P=0.15)and the depth of gastric cancer invasion(x2=1.34,P=0.55).The expression of Semaphorin 5A in cell lines SGC7901 and MKN-45 was significantly higher than that of cell lines SNU-1 and AGS(P＜0.01).Semaphorin 5A gene silencing significantly suppressed the adhesion,migration and invasion abilities of gastric cancer cells.Conclusion Semaphorin 5A may play a catalytic role in the invasion and metastasis of gastric cancer through increasing the adhesion,migration and invasion abilities of gastric cancer cell.

Objective To explore the effects of hammerhead ribozyme on the expression and activity of NHE 1 and pHi in pulmonary artery smooth muscle cells (PASMCs) of rats and its role in PASMCs proliferation in vitro . Methods According to the secondary structure of NHE 1 mRNA in rats, NHE 1 specific hammerhead ribozyme was designed with the assistance of computer. The recombinant vector of retroviral plasmid pLXSN and hammerhead ribozyme, PRZ, was transfected into the cultured PASMCs. G418 resistant cell clones were screened with 100 ?g/ml G418. Then, the expression of NHE 1 mRNA was detected by RT PCR, intracellular pH(pHi) value and recovery rate of pHi after intracellular acid loading were measured by fluorescent probe BCECF. 22 Na uptake and 3H TdR incorporation were determined, respectively. Results Compared with the cells transfected with pLXSN and non transfected cells, NHE 1 mRNA, pHi value, pHi recovery rate, 22 Na uptake and 3H TdR incorporation decreased significantly in cells transfected with recombinant vector PRZ. No significance was found between the pLXSN transfected group and non transfected group. Conclusion Hammerhead ribozyme can cleave the target NHE 1 mRNA specifically, reduce the expression of NHE 1 mRNA, induce intracellular acidosis and consequently suppress the proliferation of PASMCs.

Objective To investigate the effect of propofol on rat′s limb ischemia reperfusion injury .Methods Sixty healthy SD rats were randomly divided into sham operate group ,ischemia-reperfusion group and propofol group (n= 20) ,each group was divided into 4 subgroups according to the different reperfusion time .To copy the right lower limb ischemia reperfusion model ,5 min before reperfusion ,use propofol injection (50 mg/kg ,intraperitoneal inject) ,various subjects in the corresponding time points (3 ,6 , 9 ,12 h) were sacrificed .TNF-α ,NF-κB of blood and MDA ,SOD of Skeletal muscle were measured ,calculate muscle wet dry weight ratio .Results Compared with ischemia reperfusion group ,propofol could significantly reduce expression of TNF-alpha ,NF-κB lev-els in serum (P< 0 .05) ,inhibit the increase of the MDA level and decrease of the SOD level in muscle (P< 0 .05) ,also reduce the extent of skeletal muscle cell edema(P< 0 .05) .Conclusion Propofol can attenuate limb ischemia reperfusion injury by inhibiting inflammation response and reducing the oxygen free radicals′ damage .

Objective To evaluate the effect of activating adenylate-activated protein kinase (AMPK) on sepsis in aged mice and the relationship with autophagy.Methods Experiment [Twentyeight SPF female C57BL/6 mice,aged 16-19 months,weighing 25-35 g,were divided into sepsis group (group S,n=14) and sepsis plus AMPK agonist AICAR group (S+A group,n=14) by using a random number table method.In group S+A,AICAR 0.5 ml (dissolved in 5％ dimethyl sulfoxide) was administered by intragastric gavage.In group S,5％ dimethyl sulfoxide 0.5 ml was injected by intragastric gavage once a day for 7 consecutive days.The body weight of each mouse before and after administration was recorded.Sepsis was induced by intraperitoneal injection with cecal slurry 200 μl at the end of administration.Nine mice were selected in each group and observed for 7 days after establishing the model,and the survival rates were recorded.At 24 h after establishing the model,5 mice were sacrificed in each group,and spleen tissues were obtained for determination of the expression of AMPK,phosphorylated AMPK (p-AMPK),microtubule-associated protein 1 light chain 3 Ⅰ (LC3 Ⅰ) and LC3 Ⅱ (by Western blot).The ratios of p-AMPK/AMPK and LC3 Ⅱ/LC3 Ⅰ were calculated.Experiment Ⅱ Ten SPF female C57BL/6 mice,aged 16-19 months,weighing 25-35 g,were studied.The peritoneal macrophages obtained from 5 mice were extracted,cultured primarily and then randomized into control group (group C,n =5) and EG-FP E.Coli group (group E,n=5) using a random number table method.AICAR 0.5 ml was injected by intragastric gavage once a day for 7 consecutive days in the other 5 mice.The peritoneal macrophages were extracted after the end of intragastric administration,cultured primarily and then divided into 2 groups (n=5 each) using a random number table method:AICAR plus EGFP E.Coli group (group A+E) and AICAR plus autophagy inhibitor 3-methyladenine plus EGFP E.Coli group (group A+M+E).In group A+M+E,5 mmol/L 3-methyladenine 100 μl was added,and the cells were incubated for 1 h.EGFP expressingE.Coli was then added,and the cells were incubated for 1 h in E,A+E and A+E+M groups.Flow cytometry was used to detect the phagocytic ability of macrophages.Results Experiment Ⅰ The body weight was significantly lower after the end of administration than before administration in group S+A (P＜0.05).Compared with group S,the body weight was significantly decreased at the end of administration,the survival rate was increased at 7 days after establishing the model,the expression of LC3 Ⅱ in spleen tissues was up-regulated and the ratios of p-AMPK/AMPK and LC3 Ⅱ/LC3 Ⅰ were increased in group S+A (P＜0.05).Experiment Ⅱ Compared with group C,the phagocytic ability of macrophages was significantly enhanced in the other three groups (P＜0.05).Compared with group E,the phagocytic ability of macrophages was significantly enhanced in group A+E (P＜0.05),and no significant change was found in the phagocytic ability of macrophages in group A+M+E (P＞0.05).Compared with group A+E,the phagocytic ability of macrophages was significantly weakened in group A+M +E (P＜0.05).Conclusion Activating AMPK can increase the survival rate of aged mice with sepsis,and the mechanism is associated with enhancing autophagy of macrophages.

Objective:To explore the clinical and endoscopic features of patients with autoimmune gastritis (AIG) and to improve the accuracy of clinical diagnosis of AIG.Methods:From January 3, 2020 to November 25, 2021, the general information (gender, age), laboratory examination indicators and endoscopic findings of 179 AIG patients diagnosed at the Second Affiliated Hospital of Chongqing Medical University were retrospectively analyzed. The laboratory examination indicators included hemoglobin, gastrin-17, pepsinogen (PG), anemia combination indicators (ferritin, vitamin B 12), thyroid function indicators (thyroid-stimulating hormone, thyroglobulin antibody and thyroid peroxidase antibody), Helicobacter pylori, and anti-parietal cell antibody and anti-intrinsic factor antibody. Descriptive methods were used for statistical analysis. Results:Among the 179 AIG patients, there were 42 males (23.5%) and 137 females (76.5%), with an average age of (55.23±12.04) years old. The gastrin-17 level of AIG patients was 195.31 ng/L (143.64 ng/L, 273.61 ng/L), PG Ⅰ level and PG Ⅰ/PG Ⅱ ratio were 12.40 μg/L (7.65 μg/L, 19.40 μg/L) and 1.03 (0.66, 1.52), respectively. There were 15.3% (18/118) of the AIG patients with iron deficiency anemia, and 16.1% (19/118) with megaloblastic anemia. The positive rate of anti-parietal cell antibody was 71.8% (51/71), and the positive rate of anti-intrinsic factor antibody was 25.4% (18/71). The serum thyroid-stimulating hormone level increased in 27.3% (15/55) of the patients, and the positive rates of thyroglobulin antibody and thyroid peroxidase antibody were 31.6% (12/38) and 47.4% (18/38), respectively. The positive rate of Helicobacter pylori was 29.7% (38/128). The endoscopic appearance of AIG indicated reverse atrophy, characterized by obvious atrophy in gastric fundus and gastric body mucosa, however the atrophy of gastric antrum was not obvious. Under endoscopy yellow-white turbid mucus, which was difficult to be washed, was found in 67.0% (120/179) of the patients, and under endoscopy the residual gastric fundus glands could be seen in 19.6% (35/179) of the patients. Among 179 AIG patients, 7 cases (3.9%) of neuroendocrine tumor (NET), 7 cases (3.9%) of early gastric adenocarcinoma (including 1 case of poorly differentiated adenocarcinoma), 1 case (0.6%) of adenoma, and 14 cases (7.8%) of hyperplastic polyps were found. Except for the case of poorly differentiated adenocarcinoma undergoing surgery, the others were treated with endoscopic resection. Conclusions:When unexplained iron deficiency anemia, megaloblastic anemia, or reverse atrophy is found, AIG should be considered. AIG patients are at high risk for gastric cancer and NET, and should be closely followed up, and active treatment should be given before anemia and neurological symptoms appear.

In this study, we attempted to establish a culture system for in vitro spermatogenesis from spermatogonial stem cells (SSCs) of Bama mini-pig. Dissociated testicular cells from 1-month-old pigs were co-cultured to mimic in vivo spermatogenesis. The testicular cells were seeded in minimum essential medium alpha (α-MEM) supplemented with Knockout serum replacement (KSR). Three-dimensional colonies formed after 10 days of culture. The colonies showed positive staining for SSC-associated markers such as UCHL1, PLZF, THY1, OCT4, Dolichos biflorus agglutinin, and alkaline phosphatase. Induction of SSCs was performed in α-MEM + KSR supplemented with retinoic acid, bone morphogenetic protein 4, activin A, follicle-stimulating hormone, or testosterone. The results showed that STRA8, DMC1, PRM1, and TNP1 were upregulated significantly in the colonies after induction compared to that in testis from 1-month-old pigs, while expression levels of those genes were significantly low compared to those in 2-month-old testis. However, upregulation of ACROSIN was not significant. Replacement of α-MEM and KSR with Iscove's modified Dulbecco's medium and fetal bovine serum did not upregulate expression of these genes significantly. These results indicate that SSCs of Bama mini-pig could undergo differentiation and develop to a post-meiotic stage in α-MEM supplemented with KSR and induction factors.
Acrosin ; Activins ; Alkaline Phosphatase ; Bone Morphogenetic Protein 4 ; Dolichos ; Follicle Stimulating Hormone ; Humans ; In Vitro Techniques ; Infant ; Infant, Newborn ; Spermatogenesis ; Stem Cells ; Swine ; Testis ; Testosterone ; Tretinoin ; Up-Regulation

Objective:To test the usefulness of PET-range verification (RV) method for proton radiation accuracy verification in poly (methyl methacrylate) (PMMA) phantom using off-line PET/CT scanning.Methods:Proton irradiation dose of 2 Gy and 4 Gy were delivered in PMMA phantom. Given the difference of clinical target volume (CTV), 7 subgroups with different depth (5.0, 7.5, 10.0, 12.5, 15.0, 17.5, 20.0 cm) were set for each dose (14 radiation plans or radiation fields). PET/CT scan was performed 10 min after irradiation of 48-221 MeV proton beam. A co-registration between CT from treatment planning system and PET/CT was performed, as well as the smoothing and normalization of PET/CT data. The region of interest (ROI) and profile lines were drawn with the Raystation PET-RV software. The predictive induced radioactivity and the measured induced radioactivity profile lines were analyzed to evaluate the Δ R50, namely, the error at the position corresponding to 50% of the maximum predictive induced radioactivity at the end of both curves. Results:The size of each ROI was 5.0 cm×5.0 cm×2.5 cm. Profile lines were evenly distributed with the interval of 3 mm, and totally 289 pairs of profile lines were drew. The 2 Gy- and 4 Gy-dose groups yielded similar mean depth errors (Δ R50 between 1 mm and -1 mm with a standard deviation <1 mm). Conclusions:The off-line PET/CT scanning of PMMA phantom reveals a good agreement between predicted and measured PET data, with error of ±1 mm. The PET-RV method can be extended to clinical cases′ verification in human body treatment with further investigation.