Objective To establish a method for acquisition, perfusion, preservation and transportation of the genetically modified pig kidneys. Methods An eight genetically modified pig was utilized as experimental subject. Prior to kidneys procurement, the health status of the pig was assessed through hematology examination, and the vascular structure of the kidneys was examined using imaging techniques. Following kidneys acquisition, the pig kidneys were perfused and subsequently packaged into the cryogenic storage container labeled "For Organ Transportation Only" for interprovincial transport after communicating the transportation process with transportation department. To evaluate pathological damage to the pig kidneys, a serious of methods were employed such as hematoxylin-eosin (HE) staining, real-time fluorescent quantitative polymerase chain reaction (RT-qPCR), terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) fluorescence staining and enzyme-linked immune absorbent assay (ELISA). Results The preoperative examination of the eight genetically modified pig showed that the serum creatinine was 73.2 μmol/L, blood urea nitrogen was 2.8 mmol/L and hemoglobin was 116 g/L, all within the normal range, indicating normal renal function. CT angiography revealed no lesions in the pig kidneys, and no dilation, stenosis or premature branching of the blood vessels. The total time of obtaining the left and right kidneys from the eight genetically modified pig was (125 ± 10) min, with a blood loss of (20 ± 2) mL. The warm ischemia times were 3 min and 7 min, respectively. The perfusion and trimming times of the left and right kidneys were 36 min and 41 min, respectively. After perfusion, both kidneys were white and moist. The cold preservation and transportation time was 8 h. HE staining showed that some glomeruli were shrunk, and the lumens of the surrounding renal tubules were slightly depressed and swollen with partial inner membrane shedding and microvacuoles formed when the kidneys were preserved for 8 h. The level of cysteinyl aspartate-specific proteinase-3 messenger RNA in the kidneys tissue gradually increased with the extension of cold preservation time after 2 h (P<0.05). TUNEL fluorescence staining showed that only a small number of cells underwent apoptosis after 8 h of cold preservation, which was not significantly different from that at 0 h (P>0.05). ELISA results showed that the contents of lactate dehydrogenase (LDH) and creatinine in the preservation solution remained relatively stable, but the content of kidney injury molecule 1 (KIM-1) gradually increased with the extension of preservation time, suggesting that the pig kidneys had mild injury. Conclusions By establishing methods for acquisition, perfusion, preservation and transportation of the kidneys from genetically modified donor pig, it is possible to effectively and reliably use genetically modified pig kidneys for xenotransplantation.

Objective To explore the construction of α-1,3-galactosyltransferase (GGTA1) gene-knockout (GTKO) Diannan miniature pigs and the kidney xenotransplantation from pigs to rhesus macaques, and to assess the effectiveness of GTKO pigs. Methods The GTKO Diannan miniature pigs were constructed using the CRISPR/Cas9 gene-editing system and somatic cell cloning technology. The phenotype of GTKO pigs was verified through polymerase chain reaction, Sanger sequencing and immunofluorescence staining. Flow cytometry was used to detect antigen-antibody (IgM) binding and complement-dependent cytotoxicity. Kidney xenotransplantation was performed from GTKO pigs to rhesus macaques. The humoral immunity, cellular immunity, coagulation and physiological indicators of the recipient monkeys were monitored. The function and pathological changes of the transplanted kidneys were analyzed using ultrasonography, hematoxylin-eosin staining, immunohistochemical staining and immunofluorescence staining. Results Single-guide RNA (sgRNA) targeting exon 4 of the GGTA1 gene in Diannan miniature pigs was designed. The pGL3-GGTA1-sgRNA1-GFP vector was transfected into fetal fibroblasts of Diannan miniature pigs. After puromycin selection, two cell clones, C59# and C89#, were identified as GGTA1 gene-knockout clones. These clones were expanded to form cell lines, which were used as donor cells for somatic cell nuclear transfer. The reconstructed embryos were transferred into the oviducts of trihybrid surrogate sows, resulting in 13 fetal pigs. Among them, fetuses F04 and F11 exhibited biallelic mutations in the GGTA1 gene, and F04 had a normal karyotype. Using this GTKO fetal pig for recloning and transferring the reconstructed embryos into the oviducts of trihybrid surrogate sows, seven surviving piglets were obtained, all of which did not express α-Gal epitope. The binding of IgM from the serum of rhesus monkey 20# to GTKO pig PBMC was reduced, and the survival rate of GTKO pig PBMC in the complement-dependent cytotoxicity assay was higher than that of wild-type pig. GTKO pig kidneys were harvested and perfused until completely white. After the left kidney of the recipient monkey was removed, the pig kidney was heterotopically transplanted. Following vascular anastomosis and blood flow restoration, the pig kidney rapidly turned pink without hyperacute rejection (HAR). Urine appeared in the ureter 6 minutes later, indicating successful kidney transplantation. The right kidney of the recipient was then removed. Seven days after transplantation, the transplanted kidney had good blood flow, the recipient monkey's serum creatinine level was stable, and serum potassium and cystatin C levels were effectively controlled, although they increased 10 days after transplantation. Seven days after transplantation, the levels of white blood cells, lymphocytes, monocytes and eosinophils in the recipient monkey increased, while platelet count and fibrinogen levels decreased. The activated partial thromboplastin time, thrombin time and prothrombin time remained relatively stable but later showed an upward trend. The recipient monkey survived for 10 days. At autopsy, the transplanted kidney was found to be congested, swollen and necrotic, with a small amount of IgG deposition in the renal tissue, and a large amount of IgM, complement C3c and C4d deposition, as well as CD68⁺ macrophage infiltration. Conclusions The kidneys of GTKO Diannan miniature pigs may maintain normal renal function for a certain period in rhesus macaques and effectively overcome HAR, confirming the effectiveness of GTKO pigs for xenotransplantation.

Objective To construct a Type 1 diabetes model in miniature pigs and explore postoperative care strategies for effectively prolonging the survival time of the model pigs.Methods Seven Banna miniature pigs were selected for pancreatectomy.Glucose,vitamins,and antibiotics were administered for 3-5 days after surgery to aid recovery.Blood glucose and urine glucose levels were measured twice a day in the morning and evening to adjust insulin supplementation accordingly.The model pigs were observed daily and records were kept,including orexis,psychosis,weakness,skin ulcer,and feces and urine.Body weight was measured weekly until the death of the model animals.Based on the model pigs'condition,glucose injection and Ringer's lactate solution were administered to supplement nutrition and correct electrolyte imbalances.Results All seven Banna miniature pigs showed typical symptoms of diabetes:random blood glucose levels higher than 11.1 mmol/L after pancreatectomy,far exceeding the average blood glucose level of 6.0 mmol/L in normal pigs;positive urine glucose;and progressive weight loss.These features indicated the successful construction of Type 1 diabetes model.Additionally,Type 1 diabetic pigs that survived more than 8 weeks showed progressive hair loss and skin ulceration.Euthanasia was performed on model pigs when they were unable to stand or even eat independently,and pathological examination and HE staining were conducted on tissues collected from affected organs such as the liver,kidneys,and skin.Pathological sections revealed liver congestion,massive glycogen accumulation,ballooning degeneration of hepatocytes,and progressive liver fibrosis,along with glomerular congestion,vacuolar degeneration in renal tubular epithelial cells,proteinuria,dermal congestion,thinning of vascular walls,and varying degrees of parakeratosis and dyskeratosis in the liver,kidneys,and skin tissues due to prolonged hyperglycemia.The average survival time of the constructed Banna miniature pig diabetes model was 44 d,with a maximum survival time of 121 d.Conclusion Type 1 diabetes model can be constructed successfully in Banna miniature pigs through pancreatectomy.With meticulous postoperative care,a long-term Type 1 diabetes model with significant complications can be achieved,providing a stable large-animal model for Type 1 diabetes treatment strategies.

Objective To explore the role of zinc finger protein 36 (ZFP36) in regulating osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) and preosteoblasts. Methods ZFP36 expression was observed in primary mouse BMSCs and mouse preosteoblasts (MC3T3-E1 cells) during induced osteogenic differentiation. Zfp36-deficient cell models were constructed in the two cells using RNA interference technique and the changes in differentiation capacities of the transfected cells into osteoblasts were observed. Transcriptome sequencing was used to investigate the potential mechanisms of ZFP36 for regulating osteoblast differentiation of the two cells. U0126, a ERK/MAPK signal suppressor, was used to verify the regulatory mechanism of Zfp36 in osteogenic differentiation of Zfp36-deficient cells. Results During the 14-day induction of osteogenic differentiation, both mouse BMSCs and MC3T3-E1 cells exhibited increased expression of ZFP36, and its mRNA expression reached the peak level on Day 7 (P<0.0001). The Zfp36-deficient cell models showed reduced intensity of alkaline phosphatase (ALP) staining and alizarin red staining with significantly lowered expressions of the osteogenic marker genes including Alpl, Sp7, Bglap and Ibsp (P<0.01). Transcriptome sequencing verified the reduction of bone mineralization-related gene expressions in Zfp36-deficient cells and indicated the involvement of ERK signaling in the potential regulatory mechanism of Zfp36. Immunoblotting showed that pERK protein expression increased significantly in Zfp36-deficient cells compared with the control cells. In Zfp36-deficient MC3T3-E1 cells, inhibition of activated ERK/MAPK signaling with U0126 resulted in obviously enhanced ALP staining and significantly increased expressions of osteoblast differentiation markers Runx2 and Bglap (P<0.05). Conclusions ZFP36 is involved in the regulation of osteoblast differentiation of mouse BMSCs and preosteoblasts, and ZFP36 deficiency causes inhibition of osteoblast differentiation of the cells by activating the ERK/MAPK signaling pathway.

Objective To explore the role of zinc finger protein 36 (ZFP36) in regulating osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) and preosteoblasts. Methods ZFP36 expression was observed in primary mouse BMSCs and mouse preosteoblasts (MC3T3-E1 cells) during induced osteogenic differentiation. Zfp36-deficient cell models were constructed in the two cells using RNA interference technique and the changes in differentiation capacities of the transfected cells into osteoblasts were observed. Transcriptome sequencing was used to investigate the potential mechanisms of ZFP36 for regulating osteoblast differentiation of the two cells. U0126, a ERK/MAPK signal suppressor, was used to verify the regulatory mechanism of Zfp36 in osteogenic differentiation of Zfp36-deficient cells. Results During the 14-day induction of osteogenic differentiation, both mouse BMSCs and MC3T3-E1 cells exhibited increased expression of ZFP36, and its mRNA expression reached the peak level on Day 7 (P<0.0001). The Zfp36-deficient cell models showed reduced intensity of alkaline phosphatase (ALP) staining and alizarin red staining with significantly lowered expressions of the osteogenic marker genes including Alpl, Sp7, Bglap and Ibsp (P<0.01). Transcriptome sequencing verified the reduction of bone mineralization-related gene expressions in Zfp36-deficient cells and indicated the involvement of ERK signaling in the potential regulatory mechanism of Zfp36. Immunoblotting showed that pERK protein expression increased significantly in Zfp36-deficient cells compared with the control cells. In Zfp36-deficient MC3T3-E1 cells, inhibition of activated ERK/MAPK signaling with U0126 resulted in obviously enhanced ALP staining and significantly increased expressions of osteoblast differentiation markers Runx2 and Bglap (P<0.05). Conclusions ZFP36 is involved in the regulation of osteoblast differentiation of mouse BMSCs and preosteoblasts, and ZFP36 deficiency causes inhibition of osteoblast differentiation of the cells by activating the ERK/MAPK signaling pathway.

Objective:To investigate the relationship between orthostatic intolerance (OI) and body mass index (BMI), blood lipid and serum protein levels in children and adolescents.Methods:A total of 122 children and adolescents aged from 6 to 17 years old, who were diagnosed with OI at the Department of Pediatric Cardiology, the Second Hospital of Lanzhou University from April 2018 to April 2019, were selected as the subjects.While, 56 children and adolescents in the health management center were selected as the healthy control group during the same period.Subjects were divided into syncope group and non-syncope group according to whether there was syncope in clinical history.The height and body mass of all children were measured, and venous blood were taken to detect blood lipids and serum protein in the morning.Date analysis were conducted with SPSS 22.0 software.Results:(1) The level of triglyceride in the OI group was lower than that in the healthy control group[(0.98±0.45) mmol/L vs. (1.28±1.04) mmol/L], and there was statistically significant( t=2.025, P<0.05); the BMI were respectively (17.56±3.23) kg/m 2 and (16.46±2.58) kg/m 2 in syncope group and non-syncope group, whose result indicated that the BMI in syncope group was higher than that in non-syncope group( t=2.085, P<0.05). (2) The results of binary Logistic regression analysis showed that the triglyceride level was an independent risk factor for OI( OR=0.504, 95% CI: 0.272-0.931, P<0.05). (3) The receiver operating characteristic curve evaluated the predictive value of triacylgly-cerol to OI.Results showed the sensitivity and specificity of OI were respectively 72.1% and 48.2%when the triacylglycerol was 1.09 mmol/L. Conclusions:Low triglyceride level and high BMI may be susceptible factors to OI in children and adolescents.Therefore, the diet of children with OI should be highly valued by clinicians and parents.

Objective:To establish a mice model of inflammatory bowel disease (IBD) induced by dextran sulfate sodium (DSS), and to analyze the changes in intestinal inflammation and macrophage subsets at different stages, so as to find a new target for the treatment of IBD.Methods:Thirty male C57BL/6 mice of 6-8 weeks were randomly divided into control group, activation stage group and resolution stage group.The latter 2 groups were given 25 g/L DSS for 5 consecutive days to establish the IBD model.After 5 days, the mice were given filtered and sterilized water and sacrificed on the 10 th and 15 th day, respectively.Colon inflammation in mice was evaluated, including body weight, disease activity index (DAI) score, changes in colon length, histopathology and histopathological score.Then the expression levels of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β in colon tissues were detected by quantitative real-time PCR(qPCR). Finally, the changes of intestinal macrophage subsets were detected by flow cytometry. Results:The colon inflammation of mice in the activation stage group was significantly more severe than that in the control group, while the colon inflammation of mice in the resolution stage group was reduced.The colon length of mice in the activation stage group was (5.94±0.40) cm, which was significantly shorter than that in the control group [(7.25±0.29) cm], and the situation was slightly improved in the resolution stage with the colon length of [(6.87±0.95) cm], and the differences were statistically significant (all P<0.05). The mRNA expression levels of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α in the activation stage were 53.40±6.58, 117.69±30.78 and 2.52±0.25, respectively, which were significantly higher than those in the control group (1.00±0.13, 1.00±0.39, 1.00±0.10); the mRNA expression levels of IL-1β, IL-6 and TNF-α in the resolution stage were 2.51±0.13, 5.43±0.51 and 1.73±0.14, respectively, which were significantly lower than those in the activation stages(all P<0.05). The expression level of anti-inflammatory cytokine TGF-β in the resolution stage was 2.41±0.17, which was significantly higher than that in the activation stage (0.94±0.12), and the diffe-rence was statistically significant ( P<0.05). During the progression of IBD, there were 3 groups of macrophages in the lamina propria of intestinal mucosa of mice, of which the number of F4/80 lowCD 64-MHCⅡ - macrophage subset with the lowest maturity increased significantly in the activation stage of IBD, accounting for (10.68±4.62)%, and it decreased and returned to the normal level in the resolution stage, accounting for (4.63±1.06)%, and the difference was statistically significant ( P<0.05). Conclusions:Macrophages play an important role in the progression of IBD, the hindrance of maturation and development may be the main cause of inflammatory injury in the activation stage of IBD, and the transformation of macrophage subsets may become a new target for the treatment of IBD.

Objective:To explore the clinical characteristics, plasma levels of hydrogen sulfide(H 2S) and the relationship between the genotype and phenotype of cardiovascular involvement in children with methylmalonic acidemia and homocystinemia. Methods:The clinical and laboratory data of 66 outpatients diagnosed with methylmalonic acidemia combined with homocystinemia in Department of Pediatrics, Peking University First Hospital from January 2014 to July 2014 were collected and analyzed respectively, and the patients were divided into 2 groups: cardiovascular involvement group (10 cases) and non-cardiovascular involvement group(56 cases). The differences in the clinical characteristics, plasma levels of H 2S and genotypes were compared between 2 groups. Results:(1) There were 45 cases of early-onset children under 1 year old, including 4 cases of cardiovascular system involvement and 41 cases of non-cardiovascular system involvement.Twenty-one cases had onset above 1 year old, including 6 cases of cardiovascular system involvement and 15 cases of non-cardiovascular system involvement. There were 44 male children, including 8 cases with cardiovascular system involvement and 36 cases without cardiovascular system involvement; 22 cases female children, including 2 cases with cardiovascular system involvement and 20 cases without cardiovascular system involvement. There was no significant difference in onset age and gender distribution between the 2 groups ( χ2=2.910, 0.368, all P>0.05). (2)In the 10 cases with cardiovascular involvement, there were 3 cases with hypertension, 2 cases with hypertension combined with pulmonary hypertension, 2 cases with mild myocardial hypertrophy, 1 case with atrial septal defect combined with pulmonary hypertension, 1 case with pulmonary hypertension, 1 case with myocardial noncompaction.Compared with the non-cardiovascular involvement group, the proportion of kidney involvement was increased and that of nervous system was decreased in cardiovascular system involvement group( χ2=20.34, 5.79, all P<0.05), the proportion of hematological system involvement between the 2 groups had no significant differences ( χ2=1.28, P>0.05). The plasma levels of hydrogen sulfide of children with cardiovascular involvement was significantly lower than that of non-cardiovascular involvement[(33.8±3.6) μmol/L vs.(39.3±5.2) μmol/L, t=-3.22, P<0.01]. (3) MMACHC gene mutation (cblC type) was identified in all 46 patients.It was found that the most common type of gene mutation was c. 80A>G in cardiovascular involvement group, while c. 609G>A was the most common type of gene mutation in non-cardiovascular involvement group. Conclusions:The clinical manifestations of children with methylmalonic acidemia and homocystinemia involving cardiovascular system are multiple and prone to multiple system involvement, especially renal involvement.A decrease in plasma hydrogen sulfide levels may be involved in the involvement of its cardiovascular system.The MMACHC gene c. 80A>G mutation is the most common genetic mutation site in children with cardiovascular involvement with methylmalonic acidemia and homocystinemia.

AIM: To explore the regulatory effect of adrenomedullin (ADM) on pulmonary oxidative stress in the rats with pulmonary hypertension induced by high blood flow.METHODS: Healthy male SD rats (n=22) were randomly divided into control group, shunt group and shunt with ADM group.Abdominal aorta and inferior vena cava shunting was produced in the rats in shunt group and shunt with ADM group.After 8 weeks, ADM (1.5 μg·kg-1·h-1) was administered into the rats in shunt with ADM group subcutaneously by mini-osmotic pump for 2 weeks.Mean pulmonary artery pressure (mPAP) was evaluated by a right cardiac catheterization procedure.The ratio of right ventricular mass to left ventricular plus interventricular septal mass [RV/(LV+SP)] and relative medial thickness (RMT) in pulmonary muscularized arteries were calculated.The content of malonaldehyde (MDA), total antioxidative capacity (T-AOC), and activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in lung tissues were detected by colorimetry.The expression of NADPH oxidase 4 (NOX4) in the lung tissue was analyzed by Western blot.RESULTS: Compared with control group, the mPAP, RV/(LV+SP) and RMT in pulmonary muscularized arteries in shunt group were all significantly increased.The content of MDA and the expression of NOX4 in the lung tissues were significantly increased.The T-AOC, and activity of SOD and GSH-Px in the lung tissues were significantly decreased.However, mPAP, RV/(LV+SP) and RMT in pulmonary muscularized arteries in shunt with ADM group were significantly decreased as compared with shunt group.Meanwhile, ADM decreased the content of MDA and the expression of NOX4 in the lung tissues, but increased the T-AOC, and activity of SOD and GSH-Px in the lung tissue of shunt rats.CONCLUSION: ADM inhibits oxidative stress response in the development of pulmonary hypertension and pulmonary vascular structural remodeling induced by high pulmonary blood flow in the rats by down-regulating the NOX4 expression and strengthening the anti-oxidation response.

Objective To observe the expression of PAX-2 and PTEN in different types of endometrial lesions, and to study their relationship with endometrial intraepithelial neoplasia (EIN). Methods 60 cases of endometrial hyperplasic lesions and 70 cases of endometrial carcinoma were enrolled. All cases were reclassified by using the diagnostic criteria of EIN, and PAX-2 and PTEN were stained to compare the difference among them. Results The deletion rates of PAX-2 in benign hyperplasia, EIN and endometrial carcinoma were 39.5 % (15/38), 72.7 % (16/22) and 78.6 % (55/70), respectively, and there was a statistical difference (χ2= 21.664, P= 0.000). The deletion rates of PTEN in benign hyperplasia, EIN and endometrial carcinoma were 47.4%(18/38), 54.5%(12/22) and 75.7%(53/70), respectively, and there was no statistical difference (χ2=2.878, P=0.411). Conclusion The staining of PAX-2 could be considered as a reliable adjuvant diagnostic method in the diagnostic criteria of EIN, however, the loss of PTEN just should be regarded as a suggestion of EIN, not a confirmed diagnostic basis.