Limitation of donor source for allograft makes the research on xenograft progress. Pig is regarded as one of the ideal donor animals. The major obstacle in xenograft is hyperacute rejection, which is caused by complements after they are activated by xenogeneic antigens combined with natural antibodies. It has been confirmed that alpha-Gal is the major target antigen, whose expression is incharged by alpha-1,3 galactosyltransferase (alpha-GT). The approaches to overcome hyperacute rejection against alpha-Gal included: immunoadsorption of xenogeneic natural antibodies, lysis of antigen by enzyme and genetic manupilation to obtain animal lack of alpha-GT. Besides alpha-Gal, there were other antigens binding to human serum antibody, such as gp65 and gp100, which was expressed on PAEC after induced by TNF, the A-like antigen. But their function was still unknown. It was debatable on the role of MHC in xenograft. Both direct and indirect pathway were involved in cellular response in xenograft.
OBJECTIVE: To isolate and characterize mesenchymal stem cells (MSCs) derived from bone marrow of Banna minipig inbred line (BMI). METHODS: BMI-MSCs was isolated from bone marrow by density gradient centrifugation and cultured in DMEM (containing 15% bovine serum) at 37 degrees C with humidified 5% CO2. These cultured stem cells were characterized in clonal growth, expression of specific markers and capability of differentiation. RESULTS: Mesenchymal stem cells were proliferative and could be expanded rapidly in vitro. Clonal growth of these cells can be observed when small amount of cells was inoculated. These cells were SH2, SH3, SH4, SB10 and SB21 positive. And it was proved that these cells possess osteo-differentiation ability, up-regulated alkaline phosphatase expression and calcium secretion after osteosupplement was added into the media for several days. CONCLUSION: Mesenchymal stem cells derived from bone marrow of BMI possess the general characters of stem cell.
OBJECTIVE The major obstacle in pig to human transplantation is acute and hyperacute rejection (HAR) triggered mainly by alpha-galactosyl residues(alpha-Gal) in donor. Since the inbred-line Banna pig(IBNP) and Wuzhishan pig (IWZSP) are highly inbred and may be the potential donor for xenotransplantation, it is important to investigate the reaction between human serum and inbred-line pig tissues as well as the distribution of alpha-Gal in these tissues. METHODS Samples from heart, liver, spleen, lung, kidney, pancreas, small intestine, thymus, skin, lymph node and blood vessels at all levels were collected from four 8 to 11-month-old male IBNPs and one IWZSP. Affinity-immunohistochemistry assays were conducted following routine procedures on paraffin sections with normal human sera of blood type A, B, O, AB and BSI-B4(alpha-Gal specific binding lectin) as the primary antibodies or affinity reagents. Sections digested by alpha-galactosidase were also examined as control. RESULTS Parallel results were obtained from these pig tissues stained against human sera and BSI-B4. There was no significant difference both in the antigens recognized by sera of different blood types or BSI-B4 and in the distribution of alpha-Gal. The best alpha-Gal positive staining was appeared in vascular endothelial cells at all levels and partial parenchyma cells. However, tissues of cartilage, peripheral nerve and muscle were negative. After digested by alpha-Galactosidase, all samples were negative against BSI-B4 and human sera except few positions that showed different staining. CONCLUSION The distribution of target antigen is similar in various tissues of the two kinds of pigs. Though alpha-Gal is the major xenoantigen in IBNP and IWZSP, there may be some unknown antigens related to pig to human transplantation. Possibly the level and distribution of antigen expression in pig tissues are not the first affair to be considered, and these pigs should be genetically modified in order to eliminate rejection in pig to human xenotransplantation.
Objective To observe the effect of continuous elastic outside distraction on the change of collagen content in female mini pig’s ni pples and their supporting tissues, and to investigate the mechanism of continuous elastic outside distraction correcting inverted ni pples. Methods Three 3-month-old female mini pigs (weighing 18.5-22.0 kg), which had 12 nipples, were employed. Four nipples of each minipig were not treated as control group (n=12), and the other nipples were continuously distracted with inverted nipple correction instruments as experimental group (n=24). The nipple specimens were harvested at 2, 4, 8, and 12 weeks after distraction and HE staining was performed to observe the change oftheir tissue structure. And saturated picric acid sirius red staining was used to observe the distribution and content of collagen types I and III, image analysis software for quantitative analysis. Results The control group had normal structure of epidermis at all time points. In experimental group, the epidermis thickened; basal cells, fibroblasts, and capillary significantly prol iferated along with the times; and the content and the density of collagen types I and III increased gradually. There were significant differences in collagen type I at 4, 8, and 12 weeks, and in collagen type III at 2, 4, 8, and 12 weeks between 2 groups (P lt; 0.01). There were significant differences in the ratio of collagen type I to III at 2 and 4 weeks between 2 groups (P lt; 0.05). Conclusion Continuous elastic outside distraction can increase the quantity of collagen types I and III in the tissue, the thickness of the dermis, and the height of the nipple, which may be one of key mechanisms of correction the inverted nipple by continuous elastic outside distraction.
Objective To explore the histological changes of bio-derived bone prepared by different methods after implantation, and to provide the scaffold material from xenogeneic animal for tissue engineering. Methods Theextremities of porcine femur were cut into 0.5 cm×0.5 cm×0.5 cm. Then they were divided into 5 groups according to different preparation methods: group A was fresh bone just repeatedly rinsed by saline; group B was degreased; group C was degreased and decalcificated; group D was degreased, acellular and decalcificated; group E wasdegreased and acellular. All the materials were implantated into femoral muscle pouch of rabbit after 25 kGy irradiation sterilization. The cell counting ofinflammatory cells and osteoclasts, HE and Masson staining, material degradation, collagen and new bone formation were observed at 2, 6, and 12 weeks postoperatively. Results The residue level of trace element in biomaterials prepared by different methods is in line with the standards. All the animals survived well. There were no tissue necrosis, fluid accumulation or inflammation at all implantation sites at each time point. The inflammatory cells counting was most in group A, and there was significant difference compared with other groups(P＜0.05). There was no significant difference in osteoclasts counting among all groups. For the index of HE and Masson staining, collagen and new bone formation, groups C and D were best, group E was better, and groups A and B were worse. Conclusion The degreased, acellular and decalcificated porcine bone is better in degradation,bone formation, and lower inflammatory reaction, it can be used better scaffold material for tissue engineered bone.
Objective To study degradation of the antigen-extracted meniscus in PBS solution with no enzyme or with different enzymes. Methods Four types of enzymes (collagenase, hyaluronidase, trypsin, papain) were used to enzymolyze the antigen-extracted meniscus and the fresh meniscus for 3, 7, 15 and 30 days (37℃). The antigenextracted meniscus and the fresh meniscus were immersed in PBS solution (37℃) for 30 days. Weight loss measurement, UV spectrophotometry, and scanning electron microscopy (SEM) were used to characterize the degraded materials. Results The two types of the materials were remarkably digested under the enzymes, especially under trypsin. The degradation curves showed that the antigen-extracted meniscus was enzymolyzed less than the fresh meniscus. The degradation products were grouped as amino, peptide, and polyose by the analysis. Both of the materials could hardly behydrolyzed in PBS solution without the enzymes. The four different enzymes had different surface morphologies under the examination of SEM. Conclusion The antigen-extracted meniscus is enzymolyzed more slowly than the fresh meniscus in vitro, and the result can be used as a guideline to the further research.
OBJECTIVE: To observe the heart anatomic and histological structure of the Banna mini-pig inbred-lined and to provide the morphological data for heart xenotransplantation and breeding transgens pig. METHODS: Ten Banna mini-pigs (12-18 months old) were affused and fixed by common coratid artery. The heart were observed and measured by gross anatomy and histology. RESULTS: There were many similarities between the Banna pig heart and the human heart in anatomy and histology. However, the following differences were observed in the Banna pig heart: 1. Azygos vein directly drew into right atrium cordis. 2. The intercalated disk of cardiac muscle was less than that of human. 3. The Purkinje’s fibre was bigger than that of human. CONCLUSION: On the morphology and histology, the structure of Banna pig heart is similar to the heart of human being. It is possible that Banna minipig heart becomes organ donors for xenotransplantation.
Objective To investigate the effect of porcine small intestinal submucosa extracellular matrix (PSISM) on the vitality and gene regulation of hepatocyte so as to lay the experimental foundation for the application of PSISM in liver tissue engineering. Methods The experiment was divided into two parts: ① BRL cells were cultured with 50, 100, and 200 μg/mL PSISM-medium which were prepared by adding PSISM into the H-DMEM-medium containing 10%FBS in groups A1, B1, and C1, and simple H-DMEM-medium served as a control (group D1); ② BRL cells were seeded on 1%, 2%, and 3% PSISM hydrogel which were prepared by dissolving PSISM in sterile PBS solution containing 0.1 mol/L NaOH in groups A2, B2, and C2, and collagen type I gel served as a control (group D2). At 1, 3, and 5 days after culture, the morphology and survival of liver cells were detected by the Live/Dead fluorescent staining. The cell vitality was tested by cell counting kit-8 (CCK-8) assay. And the relative expressions of albumin (ALB), cytokeratin 18 (CK18), and alpha-fetoprotein (AFP) in hepatocytes were determined by real-time fluorescent quantitative PCR (RT-qPCR). Results The Live/Dead fluorescent staining showed the cells survived well in all groups. CCK-8 results displayed that the absorbance (A) value of group C1 was significantly higher than that of group D1 at 5 days after culture with PSISM-medium, and there was no significant difference between groups at other time points (P>0.05). After cultured with PSISM hydrogels, theA values of groups A2, B2, and C2 were significantly higher than those of group D2 at 3 and 5 days (P<0.05), theA value of group A2 was significantly higher than that of groups B2 and C2 at 5 days (P<0.05), but there was no significant difference between groups at other time points (P>0.05). RT-qPCR showed that the relative expressions of ALB and CK18 mRNA significantly increased and the relative expression of AFP mRNA significantly decreased in groups A1, B1, and C1 when compared with group D1 (P<0.05). The relative expression of CK18 mRNA in group C1 was significantly lower than that in groups A1 and B1 (P<0.05). The relative expressions of ALB and CK18 mRNA were significantly higher and the relative expression of AFP mRNA was significantly lower in groups A2, B2, and C2 than group D2 (P<0.05); the relative expression of CK18 mRNA in group A2 was significantly higher than that in group B2 (P<0.05), and the relative expression of AFP mRNA in group A2 was significantly lower than that in group C2 (P<0.05), but no significant difference was found between other groups (P>0.05). Conclusion PSISM has good compatibility with hepatocyte and can promote the vitality and functional gene expression of hepatocyte. PSISM is expected to be used as culture medium supplement or cell carrier for liver tissue engineering.
ObjectiveTo determine the optimizing parameters in transfecting the SV-40-PED cells mediated by oligofectamine. Methods With a change of Decoy oligodeoxynucleotides(ODNs)/oligofectamine in ratio and the transfection time, the uptake rate and the mean fluorescence intensity of SP1 ODNs in the SV-40-PED cells were measured by flow cytometry to evaluate the transfection efficiencies. 4 μl oligofectamine with different concentrations of ODNs(2.5，5.0，7.5，10.0 and 12.5 μl) were put into 100 μl of DMEM without serum and antibiotics. the (SV-40-PED) cells were transfected after 20 min at room temperature. the final concentration of SP1 decay ODNs were 50，100，150，200 and 250 nmol/L. Transfection effieiency was detected at 26 h after transfection. The intracellular distribution ofSP1 ODNs was determined with a fluorescence microscope. The lactate dehydrogenase (LDH) activity in the supernatant was measured to assess the cytotoxicity.Results The uptake of SP1 ODNs into the SV-40-PED cells was significantly improved by oligofectamine. The cell appearance did not change much in the groups of 50, 100 and 150 nmol/L. In the groups of 200 and 250 nmol/L, the cell reverted after being shrinked and altered to round. At 26 h after the transfection, there was no marked change in the cell form at the concentration of 250 nmol/L. There was floatation at 48 and 72 h after the transfection. Under the fluorescence microscope, we observed fluorescent materials distributed in the cell nucleus in the successfully-transferred groups. We could see the nucleoli clearly in the groups of 200 nmol/L and 250 nmol/L. There was a ber fluorescence intensitywith a higher concentration and the fluorescent materials gathered at the cell nucleus. At the final concentration of 250 nmol/L, the LDH level was 137.12±3.92 U/L in the 72hgroup, which was significantly higher those that in the 26h group（49.61±17.13 U/L）and the 48h group（120.26±8.42 U/L）(Plt;0.01). At 26 h after the transfection, there were no statistical differences at the above LDHlevels in the different-concentration groups（Pgt;0.05）. Conclusion Transfection efficiency is the highest when the final concentration of the SP1 decoy ODNs is 250 nmol/L during the incubation of for 24 h in transfecting the SV-40-PED cells.
ObjectiveTo perfect the surgical process that trachea could be reconstructed by nickel-titanium (Ni-Ti) alloy stent wrapped with autologous pericardium, and to evaluate the effectiveness and observe the complications. MethodsIn the experiment, twelve healthy Bama suckling pigs with weight of 18-25 kg were selected. The pericardium was harvested to cover the Ni-Ti alloy stent. The compound artificial trachea was used to reconstruct long-segmental (6 cm) trachea defect. The effectiveness, complications, the properties, and growth rate of the new mucosa of the artificial trachea lumen were observed. ResultsOf 12 pigs, 2 died soon because of hemorrhage and infection, respectively; 7 died at 2-4 months after operation because of hyperplasia at the middle section and blockage of phlegm plug; 3 survived after 42 weeks postoperatively, but accompanied with dyspnea symptom. At 1, 2, 3, 4, and 5 months after operation, the average crawl length of the new trachea mucosa was 1, 3, 5, 7, and 10 mm, respectively; the occurrence rates of anastomotic stenosis were 0 (0/10), 0 (0/9), 0 (0/4), 33.3% (1/3), and 33.3% (1/3) respectively; and the occurrence rates of scar hyperplasia in the middle of lumen were 20% (2/10), 66.7% (6/9), 75.0% (3/4), 66.7% (2/3), and 100% (3/3), respectively. At 7 months postoperatively, the bronchoscopy examination showed that the scar in central part of artificial trachea had the trends of stagnation, softening, and narrowing, and respiratory symptom had the trend of slight ease. Hyperplasia tissue could be found in central part of artificial trachea by autopsy and was verified to be fiber cells and necrotic tissue by pathology examination. ConclusionNi-Ti alloy stent with autologous pericardium can insure that the reconstructed tracheal lumen is unobstructed, and support the trachea epithelium regeneration; the main factors of the death of the experimental animals are the lumen hyperplasia of the artificial trachea and the blockage of the secondary phlegm plug.
