Establishment of experimental model for saphenous artery perforator flap in mini pig / 中华显微外科杂志

Objective To investigate the anatomy characteristics of saphenous artery(SA) and its perforator vessels in mini pig,and to provide a new perforator flap animal model.Methods Between May,2018 and August,2018,6 7-months-old mini pigs weight 25 kilograms were sacrificed by blooding under general anesthesia.The bilateral external iliac arteries were injected with a mixture of latex-lead oxide mixture and underwent CT-scanning to provide 3-dimensional reconstruction.The origin,diameter,courses and distribution of the SA and perforators of both hind limbs were observed.Results One of the hind limbs was abandoned due to the leakage of perfusion fluid.The other 11 hind limbs were available.The femoral artery passed through the medial femoral muscle and sartorius muscle in the middle of thigh,and run between the medial femoral muscle and gracilis muscle above the knee.Below the knee,it run on the surface of the medial leg muscles.A number of small branches were distributed in the tarsal joint and calcaneus at the medial malleolus,and there were abundant communicating branches with the posterior tibial artery.The average length of SA was (14.86±0.76) mm.The outer diameters of SA at initiative,medial tibial condyle and medial malleolus were(1.73±0.15) mm,(1.50±0.12) mm and (1.30±0.13) mm,respectively.There were a total of 79 perforators which were identified,with a mean of (7.09±1.16) perforators per hind limbs.And the average outer diameter was 0.10-0.78 (0.40±0.13) mm.Conclusion The SA constantly exist with good outer diameter.The number of perforator is abundant.SA can be used as a reliable animal model for studying and training of perforator flap.

In vitro differentiation of spermatogonial stem cells using testicular cells from Guangxi Bama mini-pig

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.

Cloning, mRNA expression and protein functional bioinformatics analysis of SLA-DOA in Banna mini-pig inbred line / 中国免疫学杂志

Objective:To clone swine leukocyte antigen,class II,DO alpha (SLA-DOA) gene from Banna mini-pig inbred line (BMI) and detect its mRNA expression level in 19 important tissues.Methods:The complete eDNA sequence of SLA-DOA gene was cloned by RT-PCR method from BMI and the mRNA expression pattern in BMI important tissues was examined by semi-quantative RT-PCR method.Nucleotide and protein sequences of SIA-DOA were used to carry out bioinformatics analysis and construct the phylogenetic tree.Results:The eDNA length of BMI SLA-DOA was 1 079 bp,which encoded a protein of 250 amino acids with molecular weight (Mw) 27.81 kD,and isoelectric point (pI) 6.48.Genome structure analysis showed it localized to Sus scrofa chromosomes 7 and consisted of four exons and three introns.Semi-quantitative expression analysis showed that SLA-DOA gene expressed highly in the lymph nodes and stomach;weakly in the heart,skin and duodenum and none in other 14 tissues.Functional bioinformatics analysis indicated that SLA-DOA protein contained one signal poptide,one transmembrane region,three conserved domains,four O-GlcNAc glycosylation sites,18 potential phosphorylation sites and to be located in the cytoplasm with 94.1％ certainty.Phylogenetic analysis demonstrated that BMI (pig) had the closest relationship with cattle.Conclusion:This study have successfully cloned the SLA-DOA gene of Banna mini-pig inbred line,performed bioinformatics analysis and tissue expression profile analysis.It will provide a basis for the studies of BMI xenotransplantation.

Mensurações do bulbo ocular e cálculo do poder dióptrico da lente intraocular em miniporcos / Ocular bulb measurements and diopter power calculation of the lens in minipigs

Objetivou-se com este trabalho determinar o poder dióptrico da lente intraocular (LIO) em miniporco e as dimensões do bulbo do olho. Foram utilizados 17 miniporcos, sadios, adultos, machos e fêmeas, com peso médio de 70kg. Em todos os olhos foram realizadas a ultrassonografia modo A, a ceratometria e a medida da distância limbo a limbo. O cálculo do poder dióptrico da LIO foi obtido utilizando-se as fórmulas Haigis, Hoffer Q, SRK/T, Holladay I e Holladay II e o software Holladay IOL Consultant(r). Na comparação entre o sexo e a lateralidade do olho, não houve diferença nas variáveis biométricas e poder da LIO. A aplicação das fórmulas (Haigis, Holladay II, Holladay I, SRK/T e Hoffer Q) possibilitou o cálculo do poder da LIO. A Holladay II, fórmula que melhor individualiza o bulbo do olho do miniporco, estima valor dióptrico ao redor de 41 D. Os miniporcos têm potencial como modelo experimental em oftalmologia, relacionado ao seu menor porte e à facilidade no manejo, especialmente em experimentos de longa duração.

The aim of this study was to determine the refractive power of intraocular lens (IOL) of mini pigs and the dimensions of the eyeball. A total of 17 (34 eyes) healthy, adult, males and female animals, with average weight of 70kg were used. For every eye, A-mode ultrasound, keratometry and the measurement of limbo-to-limbo distance were conducted, all variables for calculating the refractive power of the IOL. The value was obtained using different formulas and Holladay IOL Consultant(r) Software. Additionally, the ocular measurements were compared per sex, laterality of the eye and the different formulas used in this study (Haigis, Hoffer Q, SRK / T, Holladay I and Holladay II). In the comparison between sex and laterality of the eye, there was no difference in biometric variables and power of the IOL. The application of the employed formulas (Haigis, Holladay II, Holladay I, SRK / T and Hoffer Q) allowed the IOL power calculation for this specie, and the observed value ranged between 39.58±2.15 and 46.60±2.81 diopters. Mini pigs play an important and growing role as an experimental model for study and practice of ophthalmic procedures, specially related to their smaller size and easy management in long-term experiments.

Differential regulation of senescence and in vitro differentiation by 17β-estradiol between mesenchymal stem cells derived from male and female mini-pigs

The characterization and potential of mesenchymal stem cells (MSCs) are gender dependent and estrogen influences these properties. This study demonstrated that supplementation with 17β-estradiol (E2) increases the proliferation of bone marrow-MSCs derived from male and female mini-pigs (Mp- and Fp-BMSCs) in a concentration-dependent manner, with 10(-12) M E2 suggested as the optimal dose of E2 that led to the greatest improvement in BMSCs proliferation. Supplementation of 10(-12) M E2 resulted in down-regulation of β-galactosidase activity and pro-apoptotic activity in both BMSCs, while anti-apoptotic activity was up-regulated in only Fp-BMSCs. Further, E2 increased the osteogenic ability of Fp-BMSCs. Based on these findings, optimal utilization of E2 can improve cellular senescence and apoptosis, as well as in vitro osteogenesis of BMSCs, and could therefore be useful in stem cell therapy, particularly in bone regeneration for adult females.

Therapeutic effect of topical application of curcumin during treatment of radiation burns in a mini-pig model

Curcumin protects the skin against radiation-induced epidermal damage and prevents morphological changes induced by irradiation skin, thereby maintaining the epidermal thickness and cell density of basal layers. In this study, the effects of topical curcumin treatment on radiation burns were evaluated in a mini-pig model. Histological and clinical changes were observed five weeks after radiation exposure to the back (⁶⁰Co gamma-radiation, 50 Gy). Curcumin was applied topically to irradiated skin (200 mg/cm²) twice a day for 35 days. Curcumin application decreased the epithelial desquamation after irradiation. Additionally, when compared to the vehicle-treated group, the curcumin-treated group showed reduced expression of cyclooxygenase-2 and nuclear factor-kappaB. Furthermore, irradiation prolonged healing of biopsy wounds in the exposed area, whereas curcumin treatment stimulated wound healing. These results suggest that curcumin can improve epithelial cell survival and recovery in the skin and therefore be used to treat radiation burns.

Establishment of normal reference of radiological morphology of renal artery in mini-pigs by renal angiography

Mini-pigs have been widely employed in preclinical studies to explore new therapeutic strategies for diseases of the human urinary system; however, the normal reference of the renal artery has not been clearly investigated in the mini-pig model. Therefore, we aimed to establish a normal reference of the radiological morphology of the renal artery in mini-pigs by renal angiography via catheterization of the carotid artery. The renal angiographies obtained from 15 mini-pigs were evaluated to determine the orifice from the aorta, facing direction, size and the number of branches of renal arteries. Cranio-laterally facing renal arteries with 2 distal branches were mainly observed in the renal artery of mini-pigs. Both sides of the renal artery presented symmetrical sizes; however, the right renal artery orifice from the aorta was located more cranially than the left counterpart. The results of this study will contribute to radiological diagnosis of the renal artery as well as preclinical studies of mini-pigs.

Cloning and sequence analysis of Tibet mini-pig’ s MSTN gene / 中国比较医学杂志

Objective To amplify the encoding full-length sequence of Tibet mini-pigs myostatin ( MSTN) gene and analysis the sequence by bioinformatics software.Method The RNA of liver tissues from Tibet mini-pig was extracted, and reversely transcribed into cDNA.The gene coding region sequence of myostatin gene was amplified through RT-PCR, and then the purified product of PCR was ligated with a pMD19-T and transferred into the bacterium DH5αfor replication.The positive clones were screened and sequenced. The sequence characters were analyzed by using bioinformatics method and phylogeny evolution tree was constructed with other twelve species.Results The coding redion of MSTN gene was 1128bp, and coded 375 amino acids.The amino acid homology analysis showed that the homology rate of amino acid sequence was 99%.Conclusions Molecular phylogeny evolution indicated that it had a close relation with human, dog, banna minipig, sheep, goat, cattle, horse, chimpanzee, rat, mouse except chick and zebrafish, and the most closely related with banna minipig.

Cloning and analysis of tissue expression of PGC-1αgene in Guangxi Bama mini-pigs / 中国实验动物学报

Objective To clone the coding sequence of Guangxi Bama mini-pig PGC-1αgene, and to analyze the expression of PGC-1αgene in various tissues of mini-pigs using RT-PCR and QRT-PCR techniques.Methods The PGC-1αgene coding sequence ( CDS) was amplified by PCR from the cDNA of longissimus muscle of Guangxi Bama mini-pig. The PCR products were inserted into pEASY-T5 vector, transfected E.coli, identified and sequenced.The PGC-1αgene expression in different tissues of the Bama mini-pigs was detected by RT-PCR and QRT-PCR assays.Results The PGC-1αgene CDS of Guangxi Bama mini-pig was cloned.It was 2391 bp in length.It had 99.9%homology with the reference sequence, and had two synonymous mutations that were C-A1105 and G-A1524.The expression level of PGC-1αgene was higher in the heart and kidney, followed by liver, subcutaneous fat and longissimus muscle, but the expression was not de-tected in pancreas of Guangxi Bama mini-pig.Conclusions We have successfully cloned the PGC-1αgene of Guangxi Bama mini-pig, and detected this gene expression in six tissues.The results of this study will provide a basis for studying the effect of PGC-1αon type 2 diabetes mellitus (T2DM) in Bama mini-pigs.

Establishment of a miniature pig model of type 1 diabetes by single intravenous injection of high dose streptozotocin / 中国实验动物学报

Objective To explore the feasibility of establishing mini-pig model of type 1 diabetes by single intrave-nous injection of high dose streptozotocin .Methods Eight male Zhonghua mini-pigs (22.6 ±1.8 kg) were administrated with high-dose streptozotocin (150 mg/kg) into the ear vein .Before and 10, 30, 90 minutes, 1, 3 and 7 days after adminis-tration of streptozotocin , blood samples were obtained respectively , and used to dynamically monitor the fasting blood-glucose. C peptide and insulin levels were evaluated by IVGTT test .Results Since 24 hours after STZ administration , the fasting blood-glucose level was increased significantly compared with that of pre-administration and maintained at 16.7-20.6 mmol/L, while the C peptide and insulin levels were decreased significantly .IVGTT results showed that blood sugar levels at 1 h af-ter intravenous injection of 50%glucose were much higher than 11.1 mmol/L and failed to restore to fasting glucose levels until 2 h, insulin and c-peptide did no response after injection of glucose , always kept at a trace level .Conclusion A sin-gle high-dose streptozotocin injection can be used to establish a mini-pig model of type 1 diabetes successfully .

Effect of different prone time/supine time ratios to pathology of mini-pig with acute lung injury / 中国实用护理杂志

Objective To compare the effects of different prone and supine position duration within 24-hour period on mini-pig acute lung injury (ALI)models. Methods Fifteen female mini-pigs,mean weight (30.2±3.2) kg, length (76.5±5.9) cm were anesthetized and ventilated. Hydrochloric acid (pH 1.0)was aspirated intratracheally to create ALI model. Mini-pigs were assigned randomly into three groups: p rone 8 h group, prone 8h then supine 16 h; 16 h group, prone 16 h then supine 8 h; 24 h group, prone 24 h continuously. Min-pigs were euthanasia at the end of the experiment to take out the lung, examine the tissue change and calculate the wet to dry weight ratio of lung (W/D). Sample was cut into slices and carried on hematoxylin and eosin (HE) stain which was intended to calculate injury score by semi ration score criteria. Results W/D, and injury score were lower in 16 h group and 24 h group than the 8 h group,whereas no significant differences among the three groups. Conclusions Prone position for 16 hours or longer could decrease oxygenation by decreasing inflammation in acute lung injury(ALI)animal models.

Comparison of the Cyt b Gene Sequences in Tibet Mini-Pig and Other Chinese Pigs / 中国实验动物学报

Objective To analyze the Cyt b gene sequences in Tibet mini-pigs and clarify the differences and genetic relationship with other Chinese pigs.Method The sequence of Cyt b gene was amplified from genome DNA of Tibet mini-pig,Bama miniature pigs,Guizhou xiang pigs and Wuzhishan (WZS) pigs.After sequencing,the base sequences were compared and analysed.The blood relationship tree and evolution position of Tibet mini-pig were established.Result There were 14 mutation sites between domestic pigs in China and pigs from Europe.Besides there was a significant differenee in two nucleotide site:a T→C switch in site 420 and the G→A switch in site 883 at the same time.Conclusion Chinese pigs include Bama miniature pigs,Guizhou xiang pigs and WZS pigs,have a very close blood relationship with some of Tibet mini-pigs.It has been confirmed that there is a certain genetic differentiation in the Tibet mini-pig.

A new renal artery atherosclerosis mini pig model suitable for interventional studies / 中华肾脏病杂志

Objective To establish a mini pig model suitable for interventional studies in vivo. Methods The endothelia of unilateral renal arteries in 8 purebred Chinese experimental mini pigs(CEMP)was denuded by inflated balloons after the animals were fed with high cholesterol diet for 13 weeks.The CEMP were fed with h high cholesterol diet continuously till the 40th week.The levels of blood lipid panel and creatinine were tested at week 1,14 and 40.Bilateral renal arteries were examined with intravascular uhrasonography at week 14 and 40.The vessel samples were collected at week 40 and stained with haematoxylin-eosin,Masson trichrome technique, oil O and anti-macrophage immunohistological technique. Results Significant differences of blood lipid panel and creatinine were found between week 1 and week 40.Focal ischemic renal injury could be observed pathologically.Renal arteries of CEMP were suitable for interventional procedure such as angiography and intravascular ultrasonography.Cross-sectional information of vessels could be provided clearly by intravascular ultrasonography and the intimamedian thickness of injured renal arteries was much thicker than that of non-injured ones[(0.89±0.03)mm vs (0.30±0.02)mm,P＜0.05]as evidenced by this diagnostic technique.Pathological findings demonstrated the atheroselerotic profiles of the injured renal arteries.Fibrous and fibro-fatty plaques were the main pathologic types in this CEMP model. Conclusions An animal model with renal arterial atherosclerosis mimicking the progression of atheroselerotic renovaseular disease,which is suitable for interventional procedure is established successfully.Intravascular ultasonography may have potential clinical prospect on the evaluation of atherosclerotic renovaseular disease.

A minipig based experimental teaching model for slippery pulse / 中华中医药杂志

Objective:To establish experimental teaching model for slippery pulse by mini-pig.Methods:The model of slippery pulse was established by driping low molecule dextran in vein.The normal and slippery pulse were extracted from axil artery of mini-pig by two experienced traditional Chinese physician through double blind method.Meanwile,the correlative parameters of pulse graph of axil artery such as MSAB,MSBC,HFF,HE/HB and TW were extracted through optimizational extraction method by using NX-8 multifunctional sphygmograph.Results:The pulse rate of slippery pulse of mini-pig was slightly fast than that of normal pulse.The rhythm of slippery pulse was regularity,the nger sensation was powerful,and the pulse syate was smooth.Compared with the normal pluse,the pulse graph of slippery pluse displayed a steep ascend ramus,high and narrow B wave,tiny D wave,lower E valley and obvious F wave.HB,MSAB and MSBC increased(P

Proteomics research on myocardial tissues of Chinese experimental mini-pig model with coronary heart disease / 中国药理学通报

Aim To explore the changes of protein expression pattern of myocardial tissues in mini-pig with coronary heart disease. Methods Based on the model of mini-pig with coronary heart disease,the total proteins of myocardial tissues from each group were separated by two dimensional gel electrophoresis (2-DE).Protein exeprssion pattern in normal and model group was contrasted by Image Master 2D Elite 6.01 analysis software. Then the different proteins were identified by MALDI-TOF-MS. Results Compared with normal group,14 proteins had been determined to change:13 proteins were up-regulated and 1 proteins specifically expressed in mini-pig myocardial tissues of the disease. With the search engine MASCOT,4 proteins including HSPA8 (Hsc70)protein,apolipoprotein A-Ⅳ(apoA-Ⅳ),albumin and desmin were identified.Conclusions These four proteins may play an important role in the occurrence and development of coronary heart disease. The result will be further validated by other biological methods.

Xenotransplantation model of fetal pig skin precursor tissue / 第三军医大学学报

Objective To investigate the xenotransplantation model of fetal pig skin precursor tissue and its development after transplantaion. Methods Porcine skin precursor tissue was obtained from the embryo of gestation day 56 (E56), and made into microskin or stamp skin graft. The microskin was transplanted to the dorsal wound in BALB/c nude mice, then covered with human corpse skin. The stamp skin graft was imbedded subcutaneously into the back of nude mice, and microskin was injected subcutaneously into the auricles of nude mice. Their growth and development were observed and they were examined by HE staining at 6th and 12th week after transplantation respectively. Two-sample t test was used to analyze the size of newly grown skin tissue. Results Porcine skin precursor tissue graft in three models above survived and continued growth after transplantation, and growth ability of the dorsal wound transplantation model was significantly stronger than that of the auricle model. Epidermis and hypodermis were detected in newly grown skin tissues. Hair follicles, a few of sebaceous glands, but no sweat glands were observed in auricle model, while many sebaceous glands and sweat glands were observed in the dorsal wound model. Conclusion Transplantation of microskin to dorsal wound is the optimal model of investigating the xenotransplantation of fetal pig skin precursor tissue and its development after transplantion.

EFFECT OF LOW SELENIUM ON CHONDROCYTE DIFFERENTIATION AND DIFFERENTIAL EXPRESSION OF COLLAGEN TYPES Ⅰ , Ⅱ AND X IN ARTICULAR CARTILAGE FROM MINI-PIGS / 药物分析学报

Objective According to the distribution of low selenium areas, low nutrition state of the residents and the affecting cartilage growth and articular cartilage of Kashin-Beck Disease(KBD),the chondrocyte differentia- tion and differential expression of collagen types Ⅰ , Ⅱ and Ⅹ in articular cartilage from Chinese mini-pigs treated with low selenium were investigated in order to gain insight into the effects of these conditions on chondrocyte differ- entiation in KBD cartilage. Mothods Eleven male juvenile mini-pigs, aged from 4 weeks to 6 weeks after birth, were divided into 3 groups. The Se content in the diet of the “low Se” group was 0. 035mg/kg diet, and 0. 175 mg/kg diet in the control. For Se-supplemented group 0. 390mg /kg diet was added. The content of Se in blood was assayed at the beginning and at the end of each experiment. Samples of articular cartilage were taken from the right femur condylus, and collagen types Ⅰ , Ⅱ and Ⅹ in articular cartilage were analyzed by immunohistochemistry and in situ hybridization. Results①All cartilage samples from juvenile mini-pigs fed with low selenium diet revealed a re- duction in type Ⅹ collagen mRNA expression in the hypertrophic chondrocytes as shown by in situ hybridization, and reduced type Ⅹ collagen deposition in the lower hypertrophic zone as shown by immunohistochemistry. ②Addition of selenium to the diet restored the type Ⅹ collagen to normal level. ③Type Ⅱ collagen was evenly distributed over the entire articular cartilage in all experimental and control groups. Type Ⅱ collagen mRNA signals were most prominent in the upper articular layer as well as in the hypertrophic zone in all groups. Type Ⅱ collagen expression was restrict- ed to the zone of endochondral ossification in all experimental groups and the control. Conclusion Low selenium has an down-regulatory role on the synthesis and deposition of collagen type Ⅹ in hypertrophic chondrocytes in articular cartilage of mini-pigs. Supplement of the low Se diet with additional Se restored the signals of collagen type Ⅹ to nor- mal levels. These findings indicate that selenium deficiency may disturb chondrocyte differentiation to hypertrophic cells in the growth plate,and worthy to be investigated further.

Influence of MSCs Transplant on Ventricle Reconstruction in Mini Pig Model of Acute Myocardial Infarction / 中国药房

OBJECTIVE: To study the influence of auto messenchymal stem cells(MSCs)transplant into the infracted area on the ventricle reconstruction in mini pig model of acute myocardial infarction. METHODS: The bone marrow of mini pig was collected and centrifuged in vitro for MSCs. Then after cultivation, transformation and labeling MSCs were injected into the ligated left anterior descending coronary artery and myocardium of mini pig,while the control group was administered with DMEM of the same dose. Doppler ultrasonography was performed separately at 3 weeks and 6 weeks. RESULTS: The left ventricular ejection fraction, shortening fraction, lateral thickening , and interventricular thickening in the experimental group were significantly improved, as compared with the control group, while the ventricular chamber in the experimental group was smaller than that in the control group. CONCLUSION: MSCs transplant can help to regenerate the myocardial issue in the infarcted area, thicken the myocardium, lessen the thinningz and cardiac muscle chamber, hence to slow down the process of ventricular reconstruction .