TZAP plays an inhibitory role in the self-renewal of porcine mesenchymal stromal cells and is implicated the regulation of premature senescence via the p53 pathway.

BACKGROUND: Mesenchymal stromal cells (MSCs) were originally characterized by the ability to differentiate into different mesenchymal lineages in vitro, and their immunomodulatory and trophic functions have recently aroused significant interest in the application of MSCs in cell-based regenerative medicine. However, a major problem in clinical practice is the replicative senescence of MSCs, which limits the cell proliferation potential of MSCs after large-scale expansion. Telomeric zinc finger-associated protein (TZAP), a novel specific telomere-binding protein, was recently found to stimulate telomere trimming and prevent excessive telomere elongation. The aim of this study was to elucidate the role of TZAP in regulating MSCs senescence, differentiation and proliferation. METHOD: Primary porcine mesenchymal stromal cells (pMSCs) were isolated from the bone marrow of Tibet minipigs by a noninvasive method in combination with frequent medium changes (FMCs). The deterioration of the pMSCs' proliferation capacity and their resultant entry into senescence were analyzed by using CCK8 and EdU incorporation assays, SA-ß-gal staining and comparisons of the expression levels of cellular senescence markers (p16INK14 and p21) in pMSC cell lines with TZAP overexpression or knockout. The effects of TZAP overexpression or knockout on the differentiation potential of pMSCs were assessed by alizarin red S staining after osteogenic induction or by oil red O staining after adipogenic induction. The effect of TZAP overexpression and the involvement of the p53 signaling pathway were evaluated by detecting changes in ARF, MDM2, P53 and P21 protein levels in pMSCs. RESULTS: TZAP levels were significantly elevated in late-passage pMSCs compared to those in early-passage pMSCs. We also observed significantly increased levels of the senescence markers p16INK4A and p21. Overexpression of TZAP reduced the differentiation potential of the cells, leading to premature senescence in early-passage pMSCs, while knockout of TZAP led to the opposite phenotype in late-passage pMSCs. Furthermore, overexpression of TZAP activated the P53 pathway (ARF-MDM2-P53-P21WAF/CDKN1A) in vitro. TZAP also downregulated the expression levels of PPARγ and Cebpα, two key modulators of adipogenesis. CONCLUSIONS: This study demonstrates that the level of TZAP is closely related to differentiation potential in pMSCs and affects cellular senescence outcomes via the p53 pathway. Therefore, attenuation of intracellular TZAP levels could be a new strategy for improving the efficiency of pMSCs in cell therapy and tissue engineering applications.

Direct conversion of pig fibroblasts to chondrocyte-like cells by c-Myc.

Unexpectedly, we found that c-Myc-expressing porcine embryonic fibroblasts (PEFs) subcutaneously implanted into nude mice formed cartilage-like tissues in vivo, while previous studies revealed the direct conversion of mouse and human somatic cells into chondrocytes by the combined use of several defined factors, including c-Myc, which prompted us to explore whether PEFs can be reprogrammed to become pig induced chondrocyte-like cells (piCLCs) via ectopic expression of c-Myc alone. In this study, c-Myc-expressing PEFs, designated piCLCs, which exhibited a significantly enhanced proliferation ability in vitro, displayed a chondrogenic phenotypes in vitro, as shown by the cell morphology, toluidine blue staining, alcian blue staining and chondrocyte marker gene expression. Additionally, piCLCs with a polygonal chondrocyte-like morphology were readily and efficiently converted from PEFs by enforced c-Myc expression within 10 days, while piCLCs maintained the chondrocytic phenotype and normal karyotype during long-term subculture. piCLC-derived single clones with a chondrogenic phenotype in vitro exhibited homogeneity in cell morphology and staining intensity compared with mixed piCLCs. Although the mixtures of cartilaginous tissues and tumorous tissues accounted for ~12% (6/51) of all xenografts (51), piCLCs generated stable, homogenous, hyaline cartilage-like tissues without tumour formation at 45 out of the 51 injected sites when subcutaneously injected into nude mice. The hyaline cartilage-like tissues remained for at least 16 weeks. Taken together, these findings demonstrate for the first time the direct induction of chondrocyte-like cells from PEFs with only c-Myc.

The diagnostic value of [18F]-FDG-PET/CT in assessment of radiation renal injury in Tibet minipigs model.

BACKGROUND: Radiation-induced kidney damage can severely affect renal function, and have a serious impact on glucose reabsorption. Fluoro-2-deoxyglucose positron emission tomography (FDG-PET) is routinely utilized for metabolic imaging of glucose utilization. In this study, we are trying to assess the diagnostic value of 18F-FDG-PET/CT on measuring hyperacute effect of total body irradiation (TBI) on the kidneys. METHODS: Forty-eight Tibet minipigs were treated by TBI of different dosages using an 8-MV X-ray linear accelerator. Whole-body 18F-FDG-PET/CT was performed at 6, 24 and 72 h followed by histologic examination, blood samples' and renal function analysis. RESULTS: The uptake of 18F-FDG was significantly different between 11/14 Gy dose groups and control group, the standard Uptake Values reached a maximal level at 72 h after 14-Gy TBI treatment. At doses over 8 Gy, histological observation showed formation of tube casts, degeneration, necrosis of tubular cells, inflammatory cell infiltration and dilatation of the mitochondria of tubule cells. Renal function analysis confirmed the changes in blood urea nitrogen and creatinine levels at various dosages and time intervals. Immunohistochemistry and western blot results indicate that the expression levels of IL-10 and TNF-α proteins were positively correlated with radiation dose up to 8 Gy. CONCLUSIONS: 18F-FDG PET/CT can reflect pathological changes in kidneys and it may be a useful tool for rapid and non-invasive assessment in cases of suspected radiation-induced kidney damage.

Abnormalities of hair structure and skin histology derived from CRISPR/Cas9-based knockout of phospholipase C-delta 1 in mice.

BACKGROUND: Hairless mice have been widely applied in skin-related researches, while hairless pigs will be an ideal model for skin-related study and other biomedical researches because of the similarity of skin structure with humans. The previous study revealed that hairlessness phenotype in nude mice is caused by insufficient expression of phospholipase C-delta 1 (PLCD1), an essential molecule downstream of Foxn1, which encouraged us to generate PLCD1-deficient pigs. In this study, we plan to firstly produce PLCD1 knockout (KO) mice by CRISPR/Cas9 technology, which will lay a solid foundation for the generation of hairless PLCD1 KO pigs. METHODS: Generation of PLCD1 sgRNAs and Cas 9 mRNA was performed as described (Shao in Nat Protoc 9:2493-2512, 2014). PLCD1-modified mice (F0) were generated via co-microinjection of PLCD1-sgRNA and Cas9 mRNA into the cytoplasm of C57BL/6J zygotes. Homozygous PLCD1-deficient mice (F1) were obtained by intercrossing of F0 mice with the similar mutation. RESULTS: PLCD1-modified mice (F0) showed progressive hair loss after birth and the genotype of CRISPR/Cas9-induced mutations in exon 2 of PLCD1 locus, suggesting the sgRNA is effective to cause mutations that lead to hair growth defect. Homozygous PLCD1-deficient mice (F1) displayed baldness in abdomen and hair sparse in dorsa. Histological abnormalities of the reduced number of hair follicles, irregularly arranged and curved hair follicles, epidermal hyperplasia and disturbed differentiation of epidermis were observed in the PLCD1-deficient mice. Moreover, the expression level of PLCD1 was significantly decreased, while the expression levels of other genes (i.e., Krt1, Krt5, Krt13, loricrin and involucrin) involved in the differentiation of hair follicle were remarkerably increased in skin tissues of PLCD1-deficient mice. CONCLUSIONS: In conclusion, we achieve PLCD1 KO mice by CRISPR/Cas9 technology, which provide a new animal model for hair development research, although homozygotes don't display completely hairless phenotype as expected.

Effect of Prepregnancy Obesity on Litter Size in Primiparous Minipigs.

Obesity is a public health problem in both developed and developing countries, and the negative effects of obesity on reproductive physiology have been highlighted recently. We evaluated the effects of porcine obesity index, sex hormones, and peptide hormones on litter size in various breeds of minipigs. Blood samples were collected from sedated 8-,10-, and 12-mo-old minipigs to measure preovulatory levels of sex hormones (follicle-stimulating hormone, luteinizing hormone, estradiol, progesterone, testosterone, and prolactin) and peptide hormones (insulin-like growth factor, glucagon, cortisol, growth hormone, free thyroxine, free triiodothyronine, insulin, and leptin). We also measured weight, abdominal circumference, neck circumference, and body length and then calculated the porcine obesity index. Data were analyzed by one-way ANOVA, and means were compared by least significance difference testing. Pearson correlation between parameters and litter size was analyzed. Prepregnancy porcine obesity index and litter size were negatively correlated in primiparous minipigs. Litter size was influenced by luteinizing hormone, estradiol, progesterone, testosterone, prolactin, follicle-stimulating hormone, cortisol, insulin-like growth factor 1, growth hormone, free thyroxine, insulin, and leptin. In conclusion, prepregnancy obesity reduces litter size in primiparous minipigs.

Generation of DKK1 transgenic Tibet minipigs by somatic cell nuclear transfer (SCNT).

Hairless mice have been widely applied in skin-related researches, while hairless pigs will be a useful model for skin-related study and other biomedical researches. Dickkopf-related protein 1 (DKK1) is inhibitor of Wnt signaling pathway. Transgenic mice expressing DKK1 transgene under control of a human keratin 14 (K14) promoter display hairless phenotype, which encouraged us to generate transgenic minipigs expressing pig DKK1 transgene under control of K14 promoter and finally achieve hairless minipigs. To generate transgenic cloned pigs, we constructed the lentiviral expression vector pERKDZG which contains two independent expression cassettes, the transcription of Tibet minipig DKK1 and EGFP genes are driven by K14 promoter, while mRFP is regulated under the control of Ef-1α promoter. Prior to generating the transgenic pig, the functionality of pERKDZG construct was verified by fluorescence assay and via checking pDKK1 expression. Subsequently, lentiviruses harboring ERKDZG transgene infected porcine embryonic fibroblasts (PEFs), followed by sorting RFP-positive PEFs by flow cytometry to obtain the purified PEFs carrying ERKDZG, designated DKK1-PEFs as donor cells used for somatic cell nuclear transfer (SCNT). Finally, we obtained 3 DKK1 transgenic cloned pigs with skin-specific expression of pDKK1 and EGFP transgenes, but unfortunately, DKK1 transgenic cloned pigs don't display hairless phenotype as expected. Taken together, we achieve DKK1 transgenic cloned pigs with skin-specific expression of pDKK1 transgene which provide a pig model for exploring DKK1 gene functions in pigs.

Improved biocompatibility of poly(lactic-co-glycolic acid) orv and poly-L-lactic acid blended with nanoparticulate amorphous calcium phosphate in vascular stent applications.

Biodegradable polymers used as vascular stent coatings and stent platforms encounter a major challenge: biocompatibility in vivo, which plays an important role in in-stent restenosis (ISR). Co-formulating amorphous calcium phosphate (ACP) into poly(lactic-co-glycolic acid) (PLGA) or poly-L-lactic acid (PLLA) was investigated to address the issue. For stent coating applications, metal stents were coated with polyethylene-co-vinyl acetate/poly-n-butyl methacrylate (PEVA/PBMA), PLGA or PLGA/ACP composites, and implanted into rat aortas for one and three months. Comparing with both PEVA/PBMA and PLGA groups after one month, the results showed that stents coated with PLGA/ACP had significantly reduced restenosis (PLGA/ACP vs. PEVA/PBMA vs. PLGA: 21.24 +/- 2.59% vs. 27.54 +/- 1.19% vs. 32.12 +/- 3.93%, P < 0.05), reduced inflammation (1.25 +/- 0.35 vs. 1.77 +/- 0.38 vs. 2.30 +/- 0.21, P < 0.05) and increased speed of re-endothelialization (1.78 +/- 0.46 vs. 1.17 +/- 0.18 vs. 1.20 +/- 0.18, P < 0.05). After three months, the PLGA/ACP group still displayed lower inflammation score (1.33 +/- 0.33 vs. 2.27 +/- 0.55, P < 0.05) and higher endothelial scores (2.33 +/- 0.33 vs. 1.20 +/- 0.18, P < 0.05) as compared with the PEVA/PBMA group. Moreover, for stent platform applications, PLLA/ACP stent tube significantly reduced the inflammatory cells infiltration in the vessel walls of rabbit iliac arteries relative to their PLLA cohort (NF-kappaB-positive cells: 23.31 +/- 2.33/mm2 vs. 9.34 +/- 1.35/mm2, P < 0.05). No systemic biochemical or pathological evidence of toxicity was found in either PLGA/ACP or PLLA/ACP. The co-formulation of ACP into PLGA and PLLA resulted in improved biocompatibility without systemic toxicity.

Single intraperitoneal injection of monocrotaline as a novel large animal model of chronic pulmonary hypertension in Tibet minipigs.

OBJECTIVE: The purpose of this study was to establish an animal model of chronic pulmonary hypertension with a single-dose intraperitoneal injection of monocrotaline (MCT) in young Tibet minipigs, so as to enable both invasive and noninvasive measurements and hence facilitate future studies. METHODS: Twenty-four minipigs (8-week-old) were randomized to receive single-dose injection of 12.0 mg/kg MCT (MCT group, n = 12) or placebo (control group, n = 12 each). On day 42, all animals were evaluated for pulmonary hypertension with conventional transthoracic echocardiography, right heart catheterization (RHC), and pathological changes. Findings of these studies were compared between the two groups. RESULTS: At echocardiography, the MCT group showed significantly higher pulmonary arterial mean pressure (PAMP) compared with the controls (P<0.001). The pulmonary valve curve showed v-shaped signals with reduction of a-waves in minipigs treated with MCT. In addition, the MCT group had longer pulmonary artery pre-ejection phases, and shorter acceleration time and ejection time. RHC revealed higher mean pulmonary arterial pressure (mPAP) in the MCT group than in the control group (P<0.01). A significant and positive correlation between the mPAP values and the PAMP values (R = 0.974, P<0.0001), and a negative correlation between the mPAP and ejection time (R = 0.680, P<0.0001) was noted. Pathology demonstrated evidence of pulmonary vascular remodeling and higer index of right ventricular hypertrophy in MCT-treated minipigs. CONCLUSION: A chronic pulmonary hypertension model can be successfully established in young minipigs at six weeks after MCT injection. These minipig models exhibited features of pulmonary arterial hypertension that can be evaluated by both invasive (RHC) and noninvasive (echocardiography) measurements, and may be used as an easy and stable tool for future studies on pulmonary hypertension.

Irradiation induced injury reduces energy metabolism in small intestine of Tibet minipigs.

BACKGROUND: The radiation-induced energy metabolism dysfunction related to injury and radiation doses is largely elusive. The purpose of this study is to investigate the early response of energy metabolism in small intestinal tissue and its correlation with pathologic lesion after total body X-ray irradiation (TBI) in Tibet minipigs. METHODS AND RESULTS: 30 Tibet minipigs were assigned into 6 groups including 5 experimental groups and one control group with 6 animals each group. The minipigs in these experimental groups were subjected to a TBI of 2, 5, 8, 11, and 14 Gy, respectively. Small intestine tissues were collected at 24 h following X-ray exposure and analyzed by histology and high performance liquid chromatography (HPLC). DNA contents in this tissue were also examined. Irradiation causes pathologic lesions and mitochondrial abnormalities. The Deoxyribonucleic acid (DNA) content-corrected and uncorrected adenosine-triphosphate (ATP) and total adenine nucleotides (TAN) were significantly reduced in a dose-dependent manner by 2-8 Gy exposure, and no further reduction was observed over 8 Gy. CONCLUSION: TBI induced injury is highly dependent on the irradiation dosage in small intestine and inversely correlates with the energy metabolism, with its reduction potentially indicating the severity of injury.

The enforced expression of c-Myc in pig fibroblasts triggers mesenchymal-epithelial transition (MET) via F-actin reorganization and RhoA/Rock pathway inactivation.

In previous studies from other labs it has been well demonstrated that the ectopic expression of c-Myc in mammary epithelial cells can induce epithelial-mesenchymal transition (EMT), whereas in our pilot experiment, epithelial-like morphological changes were unexpectedly observed in c-Myc-expressing pig fibroblasts [i.e., porcine embryonic fibroblasts (PEFs) and porcine dermal fibroblasts (PDFs)] and pig mesenchymal stem cells, suggesting that the same c-Myc gene is entitled to trigger EMT in epithelial cells and mesenchymal-epithelial transition (MET) in fibroblasts. This prompted us to characterize the existence of a MET in c-Myc-expressing PEFs and PDFs at the molecular level. qRT-PCR, immunofluorescence and western blot analysis illustrated that epithelial-like morphological changes were accompanied by the increased expression of epithelial markers [such as cell adhesion proteins (E-cadherin, α-catenin and Bves), tight junction protein occludin and cytokeratins (Krt8 and Krt18)], the reduced expression of mesenchymal markers [vimentin, fibronectin 1 (FN1), snail1, collagen family of proteins (COL1A1, COL5A2) and matrix metalloproteinase (MMP) family (MMP12 and MMP14)] and the decreased cell motility and increased cell adhesion in c-Myc-expressing PEFs and PDFs. Furthermore, the ectopic expression of c-Myc in pig fibroblasts disrupted the stress fiber network, suppressed the formation of filopodia and lamellipodia, and resulted in RhoA/Rock pathway inactivation, which finally participates in epithelial-like morphological conversion. Taken together, these findings demonstrate, for the first time, that the enforced expression of c-Myc in fibroblasts can trigger MET, to which cytoskeleton depolymerization and RhoA/Rock pathway inactivation contribute.

The diagnostic value of 18F-FDG-PET/CT in hematopoietic radiation toxicity: a Tibet minipig model.

This study was undertaken to assess the diagnostic value of 2-[(18)F]-fluoro-2-deoxy-D-glucose positron emission tomography with computed tomography ([(18)F]-FDG-PET/CT) in the detection of radiation toxicity in normal bone marrow using Tibet minipigs as a model. Eighteen Tibet minipigs were caged in aseptic rooms and randomly divided into six groups. Five groups (n = 3/group) were irradiated with single doses of 2, 5, 8, 11 and 14 Gy of total body irradiation (TBI) using an 8-MV X-ray linear accelerator. These pigs were evaluated with [(18)F]-FDG-PET/CT, and their marrow nucleated cells were counted. The data were initially collected at 6, 24 and 72 h after treatment and were then collected on Days 5-60 post-TBI at 5-day intervals. At 24 and 72 h post-TBI, marrow standardized uptake value (SUV) data showed a dose-dependent decrease in the radiation dose range from 2-8 Gy. Upon long-term observation, SUV and marrow nucleated cell number in the 11-Gy and 14-Gy groups showed a continuous and marked reduction throughout the entire time course, while Kaplan-Meier curves of survival showed low survival. In contrast, the SUVs in the 2-, 5- and 8-Gy groups showed early transient increases followed by a decline from approximately 72 h through Days 5-15 and then normalized or maintained low levels through the endpoint; marrow nucleated cell number and survival curves showed approximately the same trend and higher survival, respectively. Our findings suggest that [(18)F]-FDG-PET/CT may be helpful in quickly assessing the absorbed doses and predicting the prognosis in patients.

¹8F-FDG uptake by spleen helps rapidly predict the dose level after total body irradiation in a Tibetan minipig model.

OBJECTIVES: To investigate whether (18)F- FDG uptake can be applied in dosimetry to facilitate the rapid and accurate evaluation of individual radiation doses after a nuclear accident. METHODS: Forty-eight Tibetan minipigs were randomised into a control group (n = 3) and treatment groups (n = 45). (18)F-FDG combined positron-emission tomography and computed tomography (PET/CT) were carried out before total body irradiation (TBI) and at 6, 24 and 72 h after receiving TBI doses ranging from 1 to 11 Gy. Spleen tissues and blood samples were also collected for histological examination, apoptosis and blood analysis. RESULTS: Mean standardised uptake values (SUVs) of the spleen showed significant differences between the experimental and the control groups. Spleen SUV at 6 h post-irradiation showed significant correlation with radiation dose; Spearman's correlation coefficient was 0.97 (P < 0.01). Histological observations showed that damage to the splenic lymphocyte became more severe with an increase in the radiation dose. Moreover, apoptosis was one of the major routes of splenic lymphocyte death, which was also confirmed by flow cytometry analysis. CONCLUSIONS: In the Tibetan minipig model, radiation doses have a close relationship with the (18)F-FDG uptake of the spleen. This finding suggests that (18)F-FDG PET/CT may be useful for the rapid detection of individual radiation doses.

Effect of total body X-ray irradiation on lymph node in Tibet minipig.

The purpose of this study was to determine the time-dose-effect of total body X-ray irradiation on lymphocytes in lymph nodes and peripheral blood in Tibet minipigs. Forty-eight Tibet minipigs were assigned into 6 groups including 5 experimental groups with 9 and the control group with 3. The minipigs in experimental groups were subjected to a total body X-ray irradiation of 2, 5, 8, 11, and 14 Gy respectively. Lymph nodes and peripheral blood samples were collected at 6, 24, and 72 hours after X-ray exposure and received histological microscopy examination and apoptosis analysis. Histology observation showed that the number of lymphocytes decreased within the lymph nodes with the increase of radiation doses and exposure time. The observation of transmission electron microscopy (TEM) showed typical apoptotic cells below 11 Gy while at 14 Gy necrotic cells were dominant. The apoptotic rate of lymphocytes in the lymph nodes was positively correlated with radiation dose in the range of 2-11 Gy, and reached the maximal level (39.4 ± 2.8) at 24 hours after 11 Gy irradiation, followed by a decrease in the apoptotic rate, but still higher than that of the control group. The number of lymphocytes in the peripheral blood samples was decreased significantly by increasing of the radiation dose and exposure time. We conclude that early damage of lymphocytes by total body X-ray irradiation is dose and time dependent below 11 Gy and before 24 hours post irradiation, and that the dosage of irradiation less than 11 Gy induced apoptosis, whereas the dose at 14 Gy resulted in necrosis in lymphocytes of the lymph nodes.

[Mitochondrial DNA D-loop variation types in Tibet mini-pigs in association with the blood parameters].

OBJECTIVE: To analyze the mitochondrial DNA (mtDNA) D-loop region sequence variation in Tibet Mini-Pigs in relation to the blood parameters and provide the molecular genetic basis for developing new species of laboratory animals. METHODS: The genomic DNA was extracted from the whole blood samples of 59 Tibet mini-pigs to amplifying the mtDNA D-loop for sequence analysis. Nine physiological and nine biochemical blood parameters of Tibet mini-pigs were measured . RESULTS: Based on the variation of the tandem repeat motif, the mtDNA D-loop region of Tibet mini-pigs was classified into two types, namely type A and B with the percentage of 57.6% and 42.4%, respectively, roughly matching the 3 transform sites (305, 500, 691) at the 5' end. In the 18 blood parameters, only red blood cell count showed significant differences between types A and (P<0.01). CONCLUSION: Based on the sequence variation of the mtDNA D-loop region, Tibet mini-pigs can be divided into two types that show a significant difference in red blood cell count.

[Effect of juglone on the ultrastructure of human liver cancer BEL-7402 cells].

OBJECTIVE: To study the effect of juglone on the ultrastructure of human liver cancer BEL-7402 cells. METHODS: BEL-7402 cells were incubated in the presence of 12.5 micromol/L juglone for 24 h, and fixed in 2.5% glutaraldehyde for HE staining and Coomassie brilliant blue staining and scanning electron microscopy. RESULTS: Incubation with juglone resulted in obvious changes in the cell morphology and cytoskeletal alterations of the cells. Scanning electron microscopy revealed reduced volume of the cell bodies, dissociation of the cells, curling and malformation of the microvilli on the cell surface with rupture of the intercellular junction and enlargement of the intercellular space. The formation of apoptotic bodies was observed. Transmission electron microscopy showed expansion of the endoplasmic reticula, mitochondrial cristea disintegration, nucleolar fragmentation and formation of the apoptotic bodies after the exposure to juglone for 24 h. CONCLUSION: Juglone can cause ultrastructural changes of human liver cancer BEL-7402 cells and induce their apoptosis.

[Comparative studies of hemorheological parameters in Tibet mini-pigs, Beagle dogs and human].

OBJECTIVE: To investigate the relationship of hemorheological parameters between Tibet mini-pigs, Beagle dogs and human. METHODS: Blood samples were collected from adult Tibet mini-pigs, Beagle dogs and human to detect such hemorheological parameters as the whole blood viscosity (WBV) (high, middle, and low shear rate), PV, HCT, ESR and Fi. RESULTS: The male Tibet mini-pigs had significantly lower WBV (150, 30, 5, and 1 s(-1)) and Fi than the female mini-pigs (P<0.05, 0.01, 0.05, 0.01, and 0.01, respectively). The WBV of male Beagle dogs (150 and 1 s(-1)) was significantly lower that in than female dogs (P<0.05). The WBV of male human subjects (1 s(-1)) and HCT were significantly higher, but ESR significantly lower than those in female human subjects P<0.05, 0.01 and 0.01, respectively). WBV (1 s(-1)), PV, and ESR in Beagle dogs were significantly lower, but HCT and Fi significantly higher than those in Tibet mini-pig and human subjects (P<0.05, 0.01, 0.05, and 0.01, respectively). All the hemorheological parameters were similar between Tibet mini-pigs and human (P<0.05). CONCLUSION: The hemorheological parameters of Tibet mini-pigs are closer to those of human than those of Beagle dogs.

[Mechanism of rat sciatic nerve regeneration induced by human hair keratin].

OBJECTIVE: To evaluate the effect of human hair keratin (HHK) in peripheral nerve repair and explore the mechanism of sciatic nerve regeneration. METHODS: Rat models of sciatic nerve damage was established by creating a 10-mm gap in the sciatic nerve, which was bridged with a HHK implant. Histological examinations of the nerve tissues were performed at different time points after the surgery. RESULTS: During the period from 2 days to 2 weeks following HHK implantation, Schwann cells were found to undergo dedifferentiation and proliferate along the HHK implant. Three weeks after HHK implantation, numerous macrophages and megakaryocytes occurred around the HHK, and a large quantity of regenerated Schwann cells aligned in orderly fashion was seen between the fine filaments of partially degraded HHK, where axons and capillaries were also observed. Six weeks later, massive nerve fibers and capillaries developed around the HHK, and at 9 weeks, the HHK implant was substantially degraded and numerous regenerated nerve fibers occurred characterized by obvious epineurium and perineurium. Till 12 weeks after HHK implantation, HHK was almost completely degraded and replaced by the newly regenerated nerve fibers that had grown across the nerve defect. CONCLUSIONS: HHK is an ideal material for nerve injury repair. Apocytosis plays a key role in the differentiation process of highly differentiated Schwann cells into immature Schwann cells following nerve injury. As a protective mechanism, the axons undergo enclosure and dissociation following injuries, and the intact axons give rise to growth cones that extend fibers of growing buds to competitively bind the one or more Schwann cells, but only one such but finally develops into a complete axon. The nerve fiber barrier membrane is derived from the capillary menchymal stem cells and the outmost vascular barrier membrane. The regeneration of the Schwann cells, axons and the nerve membrane is the result of self-organization through a well synchronized and coordinated mechanism.

[Methanol extract of Celastrus orbiculatu suppresses synovial hyperplasia and cartilage erosion and degradation in rheumatoid arthritis].

OBJECTIVE: To investigate the effects of methanol extract of Celastrus orbiculatu (MECO) on synovial hyperplasia and cartilage erosion and degradation in rheumatoid arthritis (RA), and explore the possible mechanisms to provide clues for new drug development for RA treatment. METHODS: The articular synovium from patients with RA and normal articular cartilage were co-implanted into the back of severe combined immunodeficient (SCID)mice to establish the chimeric model SCID- HuRAg. Four weeks later, the mice were given MECO intragastrically at 30 mg/day, leflunomide at 500 microg/day or distilled water, respectively, for 4 consecutive weeks. After completion of the treatments, the histological scores of the grafts for synovial hyperplasia, cartilage invasion by synoviocyte and cartilage degradation around the chondrocytes were evaluated, and serum level of tumor necrosis factor-alpha (TNF-alpha) was measured with radioimmunoassay. The expression of TNF-alpha mRNA and the cell apoptosis in the synovium were detected with in situ hybridization (ISH) and TUNEL, respectively, and the results were analyzed with the image analysis system. RESULTS: The grafts survived in the mice till the end of experiment. MECO and leflunomide, in comparison with distilled water, significantly lowered the scores for synovial hyperlasia (2.00+/-0.76 and 2.25+/-0.89 vs 3.63+/-0.52), cartilage erosion (1.69+/-0.80 and 2.00+/-1.36 vs 3.75+/-0.53), cartilage degradation (1.88+/-0.83 and 2.13+/-0.83 vs 3.63+/-0.74) and serum TNF-alpha level (0.84+/-0.09 and 0.83+/-0.12 vs 0.99+/-0.11 ng/ml). Cell apoptosis of the synovium increased significantly with MECO and leflunomide treatments, but the expression of TNF-alpha mRNA in the synovium decreased significantly in MECO group. CONCLUSION: MECO can effectively suppress synovial hyperplasia and cartilage erosion and degradation SCID-HuRAg mice by reducing TNF-alpha production in the synovium and promoting synovial apoptosis. MECO can be comparable with leflunomide in their effect, but the former is more effective in suppressing TNF-alpha expression in the synovium.

[Generation of transgenic mice by intratesticular injection and electroporation in vivo].

OBJECTIVE: To evaluate the feasibility of establishing transgenic mice by means of seminiferous tubule microinjection and electroporation (EP) in vivo. METHODS: Specific pathogen-free (SPF) male Kunming mice divided into 4 groups were subjected to microinjection of two different transfection solutions labeled with enhance green fluorescent protein (EGFP) into the seminiferous tubule of the testis, and in one of the two groups receiving the identical transfection solutions, EP in vivo was performed. After two weeks, the male mice of each group were mated with SPF female Kunming mice with superovulation treatment, and PCR coupled with Southern blotting was performed for the offspring mice. RESULTS: The results of PCR suggested significant difference in the efficiency of exogenous gene integration between the 4 groups (P<0.01), among which group A achieved the greatest efficiency (45%). Southern blotting did not identify significant difference between the 4 groups (P>0.05), but still suggested the highest efficiency in group A (25%). CONCLUSION: Seminiferous tubule microinjection in conjunction with subsequent EP in vivo can remarkably enhance the integration efficiency of exogenous genes into the host genome, but this new method needs to be further tested for its potential utility in transgenic animal generation.

[Experimental study of in situ electroporation-induced testis injury in specific pathogen-free male Kunming mice].

OBJECTIVE: To observe testis injuries induced by in situ electroporation in specific pathogen-free (SPF) adult male Kunming mice. METHODS: With two sets of parameters of high and low voltages, respectively, in situ electroporation of the testis was performed in vivo by fixing the testis and epididymis of the mice between a pair of rectangular tweezer-type electrodes. Two weeks after electroporation, the mice were killed and the testis and epididymis separated and fixed with 4% paraformaldehyde after recording the weight of the testis. Routine histological sections were prepared and observed under optical microscope after HE staining. The epididymis was transferred into M16 medium for spermatozoa separation, and after dilution of the spermatozoa suspension, the spermatozoa viability was observed under optical microscope. RESULTS: Two weeks after high-voltage electroporation, examination of spermatozoa viability and microscopy of the testis sections revealed irreversible testis injury, and the testis weight was significantly reduced in comparison with that of control mice (P<0.01). Low-voltage electroporation, in contrast, only caused reversible injuries of the testis, and the male mice retained their reproductive capacity after a certain length of recovery period. The testis weight after low-voltage electroporation showed no significant difference from that of the control mice (P>0.05). CONCLUSIONS: Appropriate setting of the parameters for in vivo electroporation may avoid severe impact on the reproductive capacity of the testis in SPF male Kunming mice. This technique also provides a possibility for exogenous gene transfer into the reproductive cells.