[Effects and mechanism of human umbilical vein endothelial cells-derived exosomes on wound healing in diabetic rabbits].

Objective: The investigate the effects and mechanism of exosomes derived from human umbilical vein endothelial cells (HUVECs) on wound healing in diabetes rabbits. Methods: The experimental research methods were used. The primary vascular endothelial cells (VECs) and human skin fibroblasts (HSFs) were extracted from skin tissue around ulcer by surgical excision of two patients with diabetic ulcer (the male aged 49 years and the female aged 58 years) admitted to Xiangya Third Hospital of Central South University in June 2019. The cells were successfully identified through morphological observation and flow cytometry. The HUVEC exosomes were extracted by ultracentrifugation and identified successfully by morphological observation, particle size detection, and Western blotting detection. Twenty female 3-month-old New Zealand rabbits were taken to create one type 2 diabetic full-thickness skin defect wound respectively on both sides of the back. The wounds were divided into exosomes group and phosphate buffer solution (PBS) group and treated accordingly, with 20 wounds in each group, the time of complete tissue coverage of wound was recorded. On PID 14, hematoxylin-eosin staining or Masson staining was performed to observe angiogenesis or collagen fiber hyperplasia (n=20). The VECs and HSFs were co-cultured with HUVEC exosomes for 24 h to observe the uptake of HUVEC exosomes by the two kinds of cells. The VECs and HSFs were divided to exosome group treated with HUVEC exosomes and PBS group treated with PBS to detect the cell proliferation on 4 d of culture with cell count kit 8, to detect and calculate the cell migration rate at 24 and 48 h after scratch by scratch test, to detect the cell migration number at 24 h of culture with Transwell test, and to detect the mRNA expressions of nuclear factor-erythroid 2-related factor 2 (NRF2) and transcription activating factor 3 (ATF3) by real time fluorescence quantitative reverse transcription polymerase chain reaction. Besides, the number of vascular branches and vascular length were observed in the tube forming experiment after 12 h of culture of VECs (n=3). The VECs and HSFs were taken and divided into PBS group and exosome group treated as before, and NRF2 interference group, ATF3 interference group, and no-load interference group with corresponding gene interference. The proliferation and migration of the two kinds of cells, and angiogenesis of VECs were detected as before (n=3). Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, independent sample t test, and least significant difference test. Results: The time of complete tissue coverage of wound in exosome group was (17.9±1.9) d, which was significantly shorter than (25.2±2.3) d in PBS group (t=4.54, P<0.05). On PID14, the vascular density of wound in PBS group was significantly lower than that in exosome group (t=10.12, P<0.01), and the collagen fiber hyperplasia was less than that in exosome group. After 24 h of culture, HUVEC exosomes were successfully absorbed by VECs and HSFs. The proliferative activity of HSFs and VECs in exosome group was significantly higher than that in PBS group after 4 d of culture (with t values of 54.73 and 7.05, respectively, P<0.01). At 24 and 48 h after scratch, the migration rates of HSFs (with t values of 3.42 and 11.87, respectively, P<0.05 or P<0.01) and VECs (with t values of 21.42 and 5.49, respectively, P<0.05 or P<0.01) in exosome group were significantly higher than those in PBS group. After 24 h of culture, the migration numbers of VECs and HSFs in exosome group were significantly higher than those in PBS group (with t values of 12.31 and 16.78, respectively, P<0.01). After 12 h of culture, the mRNA expressions of NRF2 in HSFs and VECs in exosome group were significantly higher than those in PBS group (with t values of 7.52 and 5.78, respectively, P<0.05 or P<0.01), and the mRNA expressions of ATF3 were significantly lower than those in PBS group (with t values of 13.44 and 8.99, respectively, P<0.01). After 12 h of culture, the number of vascular branches of VECs in exosome group was significantly more than that in PBS group (t=17.60, P<0.01), and the vascular length was significantly longer than that in PBS group (t=77.30, P<0.01). After 4 d of culture, the proliferation activity of HSFs and VECs in NRF2 interference group was significantly lower than that in PBS group and exosome group (P<0.05 or P<0.01); the proliferation activity of HSFs and VECs in ATF3 interference group was significantly higher than that in PBS group (P<0.05 or P<0.01) and significantly lower than that in exosome group (P<0.05 or P<0.01). At 24 and 48 h after scratch, the migration rates of HSFs and VECs in ATF3 interference group were significantly higher than those in PBS group (P<0.05 or P<0.01) and significantly lower than those in exosome group (P<0.05 or P<0.01). At 24 and 48 h after scratch, the migration rates of HSFs and VECs in NRF2 interference group were significantly lower than those in PBS group and exosome group (P<0.05 or P<0.01). After 24 h of culture, the migration numbers of VECs and HSFs in ATF3 interference group were significantly more than those in PBS group (P<0.05) and significantly less than those in exosome group (P<0.05 or P<0.01); the migration numbers of VECs and HSFs in NRF2 interference group were significantly less than those in PBS group and exosome group (P<0.01). After 12 h of culture, the vascular length and number of branches of VECs in NRF2 interference group were significantly decreased compared with those in PBS group and exosome group (P<0.01); the vascular length and number of branches of VECs in ATF3 interference group were significantly increased compared with those in PBS group (P<0.01) and were significantly decreased compared with those in exosome group (P<0.01). Conclusions: HUVEC exosomes can promote the wound healing of diabetic rabbits by promoting the proliferation and migration of VECs and HSFs, and NRF2 and ATF3 are obviously affected by exosomes in this process, which are the possible targets of exosome action.

Observation of rotational coherence in an excited state of CO.

The vacuum ultraviolet (VUV) radiation is generated in the strong-field-ionized CO molecules through 2+1 resonance excitation with two-color femtosecond laser pulses. When scanning the relative delay between two pump pulses, the rotational-resolved VUV radiations show periodic oscillations lasting as long as 500 ps. Fourier analysis reveals that these oscillations correspond to rotational beat frequencies of the A2Πi state of CO+, which is the result of multi-channel interference during the resonant excitation process. High resolution of Fourier transform spectra up to 0.067cm-1 allows us to obtain the fine energy levels of the A2Πi state. The theoretical calculation is in good agreement with the experimental observation. This work reveals the rotational coherence of the ionic excited state and shows the prospect of rotational coherence spectroscopy in measuring fine structures of molecular ions.

Low expression of microRNA-21 inhibits trophoblast cell infiltration through targeting PTEN.

OBJECTIVE: We investigate whether microRNA-21 could increase the infiltration ability of trophoblast cells via regulating PTEN expression, thus participating in the occurrence and development of preeclampsia. PATIENTS AND METHODS: MicroRNA-21 expression in the placenta tissues of preeclampsia women and normal pregnant women was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The effects of microRNA-21 on cell proliferation and infiltration were examined by cell counting kit-8 (CCK-8) and transwell assay, respectively. The dual-luciferase reporter gene assay was used to determine the binding relationship between microRNA-21 and PTEN. Western blot was performed to detect PTEN and microRNA-21 in trophoblasts. RESULTS: QRT-PCR results showed that the microRNA-21 expression was significantly lower in the placenta of the preeclampsia women than those of normal pregnant women. Overexpression of microRNA-21 in HTR-8/SVneo cells had no effect on cell proliferation, but enhanced cell infiltration ability. Inhibition of microRNA-21 in trophoblasts showed the opposite effects. The results of luciferase activity assay and Western blot showed that microRNA-21 could target PTEN and downregulate its expression. Overexpression of PTEN in HTR-8/SVneo cells partially reversed the enhanced invasive ability induced by microRNA-21 overexpression. CONCLUSIONS: Low expression of microRNA-21 attenuated cell infiltration of trophoblasts via direct regulating PTEN expression.

A high speed compact microwave interferometer for density fluctuation measurements in Sino-UNIted Spherical Tokamak.

A single-channel 3 mm interferometer has been developed for plasma density diagnostics in the Sino-UNIted Spherical Tokamak (SUNIST). The extremely compact microwave interferometer utilizes one corrugated feed horn antenna for both emitting and receiving the microwave. The beam path lies on the equatorial plane so the system would not suffer from beam path deflection problems due to the symmetry of the cross section. A focusing lens group and an oblique vacuum window are carefully designed to boost the signal to noise ratio, which allows this system to show good performance even with the small-diameter central column itself as a reflector, without a concave mirror. The whole system discards the reference leg for maximum compactness, which is particularly suitable for the small-sized tokamak. An auto-correcting algorithm is developed to calculate the phase evolution, and the result displays good phase stability of the whole system. The intermediate frequency is adjustable and can reach its full potential of 2 MHz for best temporal resolution. Multiple measurements during ohmic discharges proved the interferometer's capability to track typical density fluctuations in SUNIST, which enables this system to be utilized in the study of MHD activities.

Inhibition of osteogenic differentiation of mesenchymal stem cells by copper supplementation.

OBJECTIVES: Copper has been added to scaffolds when investigating bone repair, as an agent to promote vascularization; however, little is known concerning its effect on mesenchymal stem cells (MSCs), which are considered to be the origin of osteoblasts. In this study, we have aimed to elucidate effects of copper on osteogenic differentiation of MSCs. MATERIALS AND METHODS: Rat bone marrow MSCs (rBMSCs) were used as a model. Their viability was assessed by MTT assay and Roche's CASY cell counter test and calcium deposition was evaluated by staining with alizarin red S. Fluorescent phalloidin F-actin stain was used to evaluate cytoskeletal changes, protein expressions were investigated by western blotting and mRNA levels were analysed using Q-PCR. A rat model for ectopic bone formation was used to assess effects of copper on MSCs in vivo. RESULTS: Copper supplementation resulted in inhibition of osteogenesis of rBMSCs, along with reduction in expression of a number of osteogenic genes, alkaline phosphatase activity and formation of bone nodules. Cytoskeletal changes to cells during osteogenesis was inhibited by copper supplementation. In vivo study confirmed that copper could inhibit collagen formation whilst promoting angiogenesis. CONCLUSIONS: Our study demonstrated that copper inhibited osteogenic differentiation of rBMSCs in vitro. The findings caution appropriate use of copper and have laid a foundation for further research.

Molecular cloning, polymorphisms, and association analysis of the promoter region of the STAM2 gene in Wuchuan Black cattle.

The STAM protein plays an important role in the cytokine-related JAK/STAT pathway. We selected the STAM2 gene as a candidate gene that could be linked to growth performance in analysis of a Chinese cattle breed (Wuchuan Black cattle). We examined genetic variants in the promoter region of the STAM2 gene and their associations with eight growth traits in 159 individuals. Seven SNPs, which included six new SNPs for the SNP database, were found. The core promoter region was identified with a bioinformatic software. This analysis also showed that the SNPs have a significant influence on the function and structure of the STAM2 promoter in terms of RNA secondary structure, CpG island, and transcription factor binding sites. Association analysis demonstrated that G-102A is significantly associated with withers height, heart girth, cannon circumference, chest width, and hip height in this population, which leads us to suggest that G-102A is a useful SNP marker for cattle growth performance. Animals with the genotype AA had higher mean values for withers height, cannon circumference, chest width, and hip height than those with GG and AG genotypes. This SNP of the STAM2 gene could be applied in marker-assisted selection for improving growth performance in cattle.

Flocculation properties of a bioflocculant produced by Bacillus licheniformis.

Studies of the flocculation properties of bioflocculant combined with its structure characterization are helpful to develop more effective bioflocculant. This paper reports findings of our research on the flocculation properties of the bioflocculant ZS-7 in the kaolin suspension based on its structure characterization. With the addition of 2 mg/L ZS-7 and 9 mM CaCl(2), the optimum temperature for flocculation performance of ZS-7 in the kaolin suspension is about 30°C, giving the highest flocculating activity 99.2%. Studies of the flocculation properties revealed that it was stable at 60-100°C and pH 4-10. Moreover, it could flocculate a kaolin suspension over a wide range of pH (2-12) and temperatures (4-95°C) in the presence of CaCl(2).

Stromal cell-derived factor-1 promotes bone marrow-derived cells differentiation to cardiomyocyte phenotypes in vitro.

OBJECTIVE: Recent studies have demonstrated the potential of bone marrow-derived cells (BMDC) to differentiate into cardiomyocytes. Up-regulation of stromal cell-derived factor-1 (SDF-1), a member of the chemokine CXC subfamily, mediating recruitment of BMDC has been documented in infarcted myocardium; however, it remains unknown whether SDF-1 plays a role in cardiomyogenesis of BMDC. MATERIALS AND METHODS: Adherent BMDCs were cultured with SDF-1, or specific inhibitor for PI3K, CXCR4 or Akt with SDF-1, respectively. After 2 weeks, mRNAs and proteins from BMDCs were examined. RESULTS: Two weeks after supplementation with SDF-1, either murine or human adherent BMDC cultured in vitro expressed cardiac specific mRNAs (NKX2.5, atrial natriuretic factor and heavy chain beta-myosin) and proteins (troponin I and heavy chain cardiac myosin), and expression levels were partly decreased by combined treatment of CXCR4, PI3K or Akt inhibitor, with SDF-1. CONCLUSIONS: The novel findings suggest that beyond its role in mobilization and homing of BMDC, SDF-1 can promote BMDC to give rise to cardiomyocyte phenotypes in vitro, and the SDF-1/CXCR4/PI3K/Akt pathway may be one of the molecular mechanisms regulating cardiomyogenesis.

[Cellular compatibility of three natural xenogeneic bone derived biomaterials].

OBJECTIVE: To evaluate the cellular compatibility of three natural xenogeneic bone derived biomaterials. METHODS: Three types of natural xenogeneic bone derived biomaterials were made with physical and chemical treatment, composite fully deproteinized bone(CFDB), partially deproteinized bone(PDPB) and partially decalcified bone(PDCB). Three types biomaterials were cocultured with human embryonic periosteal osteoblasts. The cell growth, attachment, cell cycle, alkaline phosphatase activity were detected to evaluate the cellular compatibility to biomaterials. RESULTS: Osteoblasts attached on all three biomaterials and grew well, the effect of three biomaterials on cell proliferation was PDCB > PDPB > CFDB. The cell cycle was not obviously affected by three biomaterials. The effect of three biomaterials on alkaline phosphatase activity of osteoblasts was PDCB > PDPB > CFDB. CONCLUSION: CFDB,PDPB,PDCB have good cellular compatibility without cytotoxic and tumorigenicity, CFDB is the best. The three biomaterials can be used as scaffold materials of bone tissue engineering.

[Proliferation properties and telomerase activity of human embryonic tendon cells transformed by ptsA58H plasmid].

OBJECTIVE: To analysis the proliferation properties and telomerase activity of human embryonic tendon cells transformed by ptsA58H plasmid cultured in vitro continuously. METHODS: The 40th, 70th, and 75th passages of transformed human embryonic tendon cells (THETC) were adopted. The collagen secretion of THETC was detected by immunohistochemical methods, the growth curve of different passages of THETC was compared, and chromosome karyotype was analyzed. Total RNA of THETC were extracted to detect human telomerase reverse transcriptase (hTERT) mRNA expression by RT-PCR technique. RESULTS: When THETC were subcultured to 70 passages, the morphological characteristics of cells changed and began replicative senescence. THETC still could secret type I collagen normally. The chromosome of THETC was heteroploid (2n = 94). There were no hTERT mRNA expression. CONCLUSION: SV40 transfection can not make human embryonic tendon cells immortalization, on the other hand, human embryonic tendon cells transformed by ptsA58H plasmid has no tendency of malignant transformation.

[The influence of tissue engineered tendon on subgroup of T lymphocytes and its receptor in roman chickens].

OBJECTIVE: To investigate the influence of tissue engineered tendon on subgroup of T lymphocytes and its receptor in Roman chickens. METHODS: The flexor digitorum profundus of the third toes of right feet in 75 Roman chickens were resected and made 2.5 cm defects as experimental model. They were randomly divided into five groups according to five repair methods: no operation (group A), autograft (group B), fresh allograft (group C), polymer combined with allogenous tendon cells (group D), derived tendon materials combined with allogenous tendon cells (group E). The proliferation and transformation of lymphocytes and contribution of CD4+, CD8+, CD28 and T cell receptor (TCR) were detected to study the immune response. RESULTS: The CD4+, CD8+ and TCR of group D and E were increased slightly than that of group B after 7 days, while after 14 days, those data decreased gradually and no significant difference between tissue engineered tendon and autografts (P > 0.05), and there was significant difference between fresh allograft and tissue engineered tendon (P < 0.05). Lymphocytes transformation induced by conA also showed no significant difference between tissue engineered tendon and autografts (P > 0.05). CONCLUSION: Tendon cells are hypoantigen cells, there are less secretion of soluble antigen or antigen chips dropped out from cells. Tissue engineered tendon has excellent biocompatibility.

[Repair of growth plate defects of rabbits with cultured cartilage transplantation].

OBJECTIVE: To prevent early closure of growth plate and developmental deformities of limbs by allografts of cultured cartilages into growth plate defects of rabbits. METHODS: Chondrocytes isolated from articular cartilage of 1-month rabbits formed cartilage after cultivation in centrifuge tubes. The cartilages cultured for two weeks were implanted into growth plate defects of proximal tibiae of 6-weeks rabbits. At 4th and 16th weeks, X-ray, histologic and immunohistochemical examination were performed. RESULTS: The tibiae had no marked deformities after 4 weeks of operation. Histologic examinations showed that the defects were filled with cartilage. Immunohistochemical results of type II collagen were positive. The tibiae with allografts of cultured cartilages had no evident deformities after 16 weeks of operation. Histologic examination showed nearly closure of growth plates. On the contrary, the tibiae on control side formed severe deformities and growth plate were closed. CONCLUSION: Allograft of cultured cartilages into growth plate defects may replace lost growth plate tissues, maintain normal growth of limbs and prevent developmental deformity.

[The proliferation and differentiation of primary human embryonic skeletal myoblasts].

OBJECTIVE: To observe the proliferation and differentiation properties of primary human embryonic skeletal myoblasts cultured in vitro. METHODS: The skeletal muscle samples were obtained from 20 to 25-week abortion fetus, the family history of inherited myopathies of parental generation was negative. With a modified method of Blau, the muscle sample was digested with trypsin and collagenase. The isolated cell suspension was a mixture of myoblasts and fibroblasts, the latter was removed by repeated attachment to culture dishes. The morphological, immunohistochemical observation, the proliferation and differentiation of primary myoblasts were studied. RESULTS: The isolated myoblasts were spherical in cell suspension and spindle-like after attached to culture dishes. The myosin specialized immunohistochemical staining was strongly positive. A large quantity of skeletal muscle specialized creatine kinase (CK-MM) was synthesized in cultured myoblasts. Additionally, while the cell density of myoblasts increased, the monocyte myoblasts would fused to form multinucleated myotube. All those indicated that the cultured cells were myoblasts. Primary myoblasts proliferated quickly, the doubling time, measured in growth curve, was 4.8 days. CONCLUSION: A large number of myoblasts can be available with digestion and repeated attachment method. The cultured cells can be proved as myoblasts by morphological and immunohistochemical detection. The cultured myoblasts have good ability of proliferation and differentiation.

Green cone opsin and rhodopsin regulation by CNTF and staurosporine in cultured chick photoreceptors.

PURPOSE: To investigate the regulation of visual pigment expression in chick embryo photoreceptor cells by ciliary neurotrophic factor (CNTF), and by the protein kinase inhibitor staurosporine. METHODS: Embryonic day (ED) 8 chick embryo retinal cells were dissociated and cultured at low densities for 3 days, either in control medium or in medium supplemented with CNTF or staurosporine. The cultures were analyzed by immunocytochemistry with the monoclonal antibody Rho4D2, which recognizes chicken rhodopsin and green cone pigment, and by reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analysis to investigate visual pigment expression at the mRNA level. RESULTS: CNTF increased the number of Rho4D2-immunoreactive photoreceptors in retinal cell cultures, in agreement with previous reports. RT-PCR and Northern blot analysis, however, showed that rhodopsin mRNA was undetectable in both control and CNTF-treated cultures but that CNTF induced significant increases in mRNA levels for the green cone pigment. Staurosporine-treated cultures also had more Rho4D2-immunoreactive cells than control cultures, but this increase was accompanied by induction of rhodopsin expression, with concomitant decreases in levels of green cone pigment mRNA. No significant differences were found between CNTF- or staurosporine-treated cultures and the corresponding control cultures regarding the red cone pigment, which was expressed in all cases, and the blue and violet pigments, which were not detected in any of the samples. CONCLUSIONS: The results suggest that multiple regulatory systems control visual pigment expression during differentiation of chick embryo photoreceptor cells. CNTF appears to stimulate specifically the differentiation of green cones, without the previously suggested effects on the differentiation of rod photoreceptors in ED 8 chick retinal cultures.

[Huge thoracic wall defect repaired by tissue engineered bone transplantation].

OBJECTIVE: A rare huge desmoplastic fibroma on thoracic wall in 1 female case of 25 years old was resected, and the accompanying huge thoracic wall defect, ribs and soft tissues were repaired by tissue engineered bone and pedicled flap. The paper aims to explore the clinical results of early stage after operation. METHODS: Autogeneic bone marrow stromal cells (MSC) were obtained from bone marrow puncture of iliac bone and isolated and cultured according to the Houghton's methods, MSC were directively induced and differentiated to osteoblasts. Allogeneic ribs were made to the bio-derived bone scaffold materials after treatment of decell, deantigen, decalcification and dry freezing. 5 x 10(6)/ml MSC were cocultured with the bio-derived bone for 6 days in vitro. After intact resection of tumor, the diaphragm flap was applied to repair pleural cavity, the three defect ribs were repaired by tissue engineered bone and the soft tissue defect was repaired by transfer of pedicled ipsilateral abdominal flaps. RESULTS: The patient recovered well with first intention. Followed up for 3 months, tissue engineered ribs were matured in vitro and the heart and pulmonary functions were improved markedly. CONCLUSION: The tissue engineered bone constructed by autogeneic MSC is advantageous in individual treatment.

[Biological characteristics of continuously subcultured human embryonic skeletal myoblasts].

OBJECTIVE: To investigate the biological characteristics of continuously subcultured human embryonic skeletal myoblasts, and choose the optimal seeding cells for muscle tissue engineering. METHODS: Human embryonic skeletal myoblasts were subcultured in vitro. The growth curve, rate of myotube formation(RMF) were used to evaluate the proliferative and differentiation ability of myoblasts, and to investigate the influence of fibroblasts contamination on myoblasts. RESULTS: The beginning 6 passages of myoblasts showed strong proliferative and differentiation ability. From the 8th to 20th passage, the rate of fibroblasts contamination was increased, it mainly showed the growth characteristics of fibroblasts with increased proliferation and low differentiation. After subcultured to the 20th passage, the degeneration of myoblasts was obvious. CONCLUSION: The myoblasts within 6 passages should be used as the seeding cells of muscle tissue engineering because of strong proliferative ability and high rate of myotube formation.

[Short tandem repeat loci examination after repair of coracoclavicular ligament injury by tissue engineered tendon].

OBJECTIVE: To evaluate the result of clinical application in the repair of coracoclavicular ligament injury by tissue engineered tendon using the technique of short tandem repeat loci examination. METHODS: In september 1999, human embryonic tendon cells and artificial materials were co-cultured in vitro to construct tissue engineered tendon, which repaired coracoclavicular ligament injury. After 6 months of operation, micro-tissue were sampled during the operation of removal of internal fixation, and morphological characteristics were examined by HE staining, DNA of tissues were extracted to examine D3S1754 and Cyar04 gene loci. RESULTS: The shoulder function of the patient was recovered well after operation, and no local or systemic immunological rejection were occurred. The electrophoresis typing showed 13/14 at D3S1754 and 8/9 at Cyar04 in the tissue of tissue engineered tendon, while the autogenous ligament were 13/13 and 8/8 at D3S1754 and Cyar04 loci respectively, which suggested that the tissue engineered tendon was survived in vivo. CONCLUSION: The examination of short tandem repeat loci is a better index to evaluate the survival situation of tissue engineered tissue after transplantation in clinical application.

[SV40 and cell immortalization].

OBJECTIVE: To explore the SV40-mediated immortalization, the related factors and their roles in cell immortalization. METHODS: The original articles about cell immortalization and replicative senescence in recent decade were reviewed. RESULTS: Cell immortalization was a multifaceted phenomenon, it was involved in viral DNA integration, activation of telomerase, inactivation of growth suppressors, and so on, and their roles were closely related. CONCLUSION: The research on cell immortalization may be expected to provide important insights into a broad range of cellular biological phenomenon, and the immortalized cells can play important roles in the research of cell engineering and tissue engineering as standard cells.

[Culture of the transformed human embryonic tendon cells and its biological characteristics in vitro].

OBJECTIVE: This paper was to study the biological characteristics of the transformed human embryonic tendon cells, the relation between cell growth and culture conditions, and to compare these features with that of human embryonic tendon cells. METHODS: The pts A58H plasmid had successfully used to transform a tendon cell line from human embryo in our past work. The human embryonic tendon cells and the transformed human embryonic tendon cells were cultured in vitro. In different culture conditions, the growth curve were drawn respectively. Population dependence and proliferation capability of the cells were investigated through plate cloning test and soft agar culture. The collagen secreted by cells was identified by immunohistochemical method. RESULTS: In routine culture condition, the growth properties of the human embryonic tendon cell and transformed cells were almost identical. The growth properties of the transformed cells were not changed when the cells were frozen storage. There were changes of growth characteristics of the transformed cells when the culture temperature was changed. The transformed cells could subcultured continually and permanently. The proliferation capability of the transformed cells were stronger than that of the human embryonic tendon cells. Moreover, the growth of the transformed cells was serum-dependent, and the phenomenon of contact inhibition was observed. The transformed cells were not able to grow on soft agar culture. They had the capacity of secreting collagen type I. CONCLUSION: The transformed human embryonic tendon cells could be subcultured continually and permanently, and their growth could be controlled by changing their culture conditions and they had no malignant tendency in biological characteristics. They could be taken as an ideal experimental material for tendon engineering.

Calcineurin inhibition prevents calpain-mediated proteolysis of tau in differentiated PC12 cells.

The effects of calcium influx on tau levels and phosphorylation were examined in differentiated PC12 cells. Maitotoxin-induced calcium influx resulted in time- and concentration-dependent tau dephosphorylation and degradation. Incubation of PC12 cells with a membrane-permeable calpain inhibitor blocked maitotoxin-induced tau degradation, suggesting the involvement of calpain in calcium-stimulated tau turnover. Okadaic acid or the calcineurin inhibitor FK520 partially inhibited maitotoxin-induced tau dephosphorylation at the Tau-1 epitope, indicating both phosphatase 2A/1 and calcineurin were involved. In addition, FK520, but not okadaic acid, blocked the maitotoxin-induced tau degradation, demonstrating that dephosphorylation of specific tau epitopes by was essential for calpain-mediated tau degradation. Moreover, maitotoxin effects were likely independent of tau association with microtubules because maitotoxin induced tau degradation and dephosphorylation in the presence of either nocodazole or taxol. These data provide evidence that calpain is involved in tau turnover in situ and calcineurin plays an important role in modulating tau susceptibility to calpain.