Bone marrow mesenchymal stem cell transplantation inhibits apoptosis in the rat spinal cord injured by acrylamide / 中国组织工程研究

BACKGROUND: Until now, there is no effective treatment for peripheral neuropathy caused by acrylamide. Therefore, it is necessary to explore new treatment methods. OBJECTIVE: To explore the protection role and its mechanism of bone marrow mesenchymal stem cells (BMSCs) against acrylamide-induced intoxication in the spinal cords of rats. METHODS: BMSCs were cultured by the whole bone marrow adherence method and identified by morphological observation and flow cytometry detection. Thirty Sprague-Dawley rats, clean grade, were randomly divided into three groups (n=10 for each group): normal control group, acrylamide group and BMSCs transplantation group. The latter two groups received acrylamide by gavage, 50 mg/(kg?d), 5 days per week, for 2 weeks with an interval of 2 days. Then, in the BMSCs transplantation group, 3×106BMSCs were transplanted by the caudal vein, 5 days per week, for 3 consecutive weeks. Hematoxylin-eosin staining was utilized to observe the morphological changes of the spinal cord. Tunel assay was used to detect cell apoptosis. Western blot assay was adopted to detect the expression levels of Bcl-2 and Bax. RESULTS AND CONCLUSION: In the acrylamide-exposed rats, the damage to the structure was found in the spinal cords by morphological observation, which was significantly alleviated after BMSCs transplantation. The disturbed expression levels of Bax and Bcl-2 were also significantly inversed after BMSCs transplantation (P < 0.05). These results suggest that BMSCs transplantation can inhibit cell apoptosis in the spinal cords of acrylamide-intoxicated rats, probably by up-regulating expression of Bcl-2 and down-regulating expression of Bax.

Therapeutic effect of bone marrow mesenchymal stem cell transplantation for spinal cord injury / 中国组织工程研究

BACKGROUND: At present, treatments for spinal cord injury are limited, with poor outcomes. Therefore, it is of great importance to explore new treatment methods. OBJECTIVE:To observe the therapeutic effect of bone marrow mesenchymal stem cells(BMSCs)injection via the caudal vein on spinal cord injury in rats. METHODS:BMSCs were isolated and cultured in vitro by whole bone marrow adherence method.The surface markers were identified by flow cytometry. Thirty Sprague-Dawley rats were randomly divided into control group, spinal cord injury group and BMSCs transplantation group, 10 rats in each group. A rat spinal cord injury model was established by occlusion of the 10ththoracic vertebra using an aneurysm clamp, and 2×106BMSCs were injected through the caudal vein at 10 minutes after modeling. Basso-Bettle-Bresnahan (BBB) score for motor function recovery was assessed at 0, 10, 20, 30 days after transplantation. The therapeutic effect was evaluated by hematoxylin-eosin staining and electron transmission microscopy at 30 days after implantation. RESULTS AND CONCLUSION: Under the microscope, fusiformis-shaped or fibroblast-like cells were observed. The expression rate of CD44 and CD90 was more than 90% and the expression of CD45 was less than 2%, by which, the BMSCs were identified. The BBB scores were significantly higher in the BMSCs transplantation group than the spinal cord injury group at 20 and 30 days after transplantation (P < 0.05). Hematoxylin-eosin staining showed that there was spinal cord tissue damage, vascular rupture injury, neuronal cell degeneration and inflammatory cell infiltration in the spinal cord injury group. After BMSCs transplantation, the number of spinal cord neurons was markedly increased with intact cytomembrane and clear nucleolus. Electron microscopic results showed that spinal cord axon swelling, demyelination, nerve axon deformation and necrosis were observed in the spinal cord injury group, while after BMSCs transplantation, the rat spinal cord axon structure was repaired,and partially lost myelin was recovered with uniform thickness.To conclude,BMSCs transplantation via the caudal vein has a significant therapeutic effect on spinal cord injury in rats by repairing the spinal cord structure and protecting the integrity of axon and myelin structures.

Levels and dynamic changes of serum fibroblast growth factor 23 in hypophosphatemic rickets/osteomalacia / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Hypophosphatemic rickets/osteomalacia is a group of diseases characterised by defective mineralization of bone due to hypophosphatemia and low 1,25-dihydroxy vitamin D. To explore the role of fibroblast growth factor 23 (FGF-23) in the regulation of phosphate homeostasis, we measured the circulating concentrations of this growth factor in healthy individuals and in patients with hypophosphatemic rickets/osteomalacia.</p><p><b>METHODS</b>Nineteen patients with hypophosphatemic rickets/osteomalacia were included in hypophosphatemic group (HP, 12 female and 7 male, mean age was 30 years), and 19 healthy age-matched individuals served as the control group. Full length FGF-23 fragments were measured by two-site enzyme-linked immunosorbent assay.</p><p><b>RESULTS</b>Mean FGF-23 concentrations were significantly higher in the HP group ((87.4 +/- 43.6) pg/ml) compared with the control group ((19.2 +/- 6.16) pg/ml; P < 0.001). In 1 patient with tumour-induced osteomalacia, serum FGF-23 concentrations were 84.1 pg/ml; these concentrations were normalized 2 hours after a hemangiopericytoma resection (7.8 pg/ml). Subsequently, serum 1,25(OH)(2) vitamin D3 concentrations significantly increased from 21.3 pg/ml to 89.3 pg/ml, and serum phosphorus levels were normalized.</p><p><b>CONCLUSIONS</b>Serum FGF-23 concentrations were markedly elevated in patients with hypophosphatemic rickets. FGF-23 plays an important role in the pathogenesis of hypophosphatemic rickets/osteomalacia.</p>

Inhibitory effect of cucurbitacin E on the proliferation of ovarian cancer cells and its mechanism / 癌症

<p><b>BACKGROUND AND OBJECTIVE</b>There are various biological activities of cucurbitacin E (CuE), including antitumor effect, anti-chemical carcino-genesis, liver protection, and enhancement of the immunity, and so on. This study was to investigate the effect of CuE on proliferation inhibition and apoptosis induction of ovarian cancer ES-2 cells, and to explore the mechanism.</p><p><b>METHODS</b>ES-2 cells were treated with different concentrations of CuE for 24, 48, and 72 h, respectively. Cell proliferation was tested by MTT assay. The morphologic changes and apoptosis were observed under inverted microscope and fluorescent microscope. Cell cycle distribution was evaluated with flow cytometry. The expression of p-STAT3 was determined by Western blot.</p><p><b>RESULTS</b>The number of ES-2 significantly decreased as the concentration of CuE increased or the time prolonged. Flow cytometry analysis showed that the ratio of ES-2 cells treated 1 micromol/L CuE for 24 h increased both in S phase [from (10.55+/-0.91)% to ( 16.31 +/- 4.61) % ] and in G(2)/M phase [from (18.53+/-1.43)% to (58.34 +/- 5.77)%], while decreased in G(1) phase [from (73.13 +/-4.70)% to (23.12 +/- 5.45)%] (P<0.05). The marked morphological changes of cell apoptosis were clearly observed in ES-2 cells treated with CuE. CuE inhibited the STAT3 phosphorylation in ES-2 cell in a dose- dependent manner.</p><p><b>CONCLUSION</b>CuE can inhibit ES-2 proliferation and induce apoptosis and cell cycle arrest, which may be related to the decreased expression of the intracellular STAT3 phosphorylation.</p>

Effect of fibroblast growth factor 9 on Runx2 gene promoter activity in MC3T3-E1 and C2C12 cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Fibroblast growth factor 9 (FGF9), expressed in brain, kidney and developing skeletal tissues, can physiologically inhibit endochondral ossification; but little is known about how FGF9 affects osteoblasts and its detailed regulatory mechanism. Here we examined the effect of FGF9 on the activity of the murine Runt-related transcription factor 2 (Runx2) gene promoter in preosteoblast MC3T3-E1 and premyoblast C2C12 cells.</p><p><b>METHODS</b>Plasmids containing the Runx2 promoter region were transfected into MC3T3-E1 and C2C12 cells and stably transfected cell lines were established. The method of luciferase reporter gene activation was used to examine the effects of FGF9 on the promoter activity.</p><p><b>RESULTS</b>FGF9 (10 ng/ml) increased Runx2 promoter activity in MC3T3-E1 cells. When MC3T3-E1 cells were treated with FGF9 plus the various inhibitors or activator of the intracellular signaling transducation pathways, including 10 micromol/L U0126 (the inhibitor of mitogen-activated protein kinase kinase), 10 micromol/L SB203580 (the inhibitor of p38/mitogen activated protein kinase), or 1 micromol/L C6 ceramide (an activator of mitogen activated protein kinase), the luciferase expression did not change significantly compared with that of the cells treated with FGF9 only. However, when C2C12 cells were treated with 10 ng/ml FGF9, Runx2 gene promoter activity first decreased and then increased over a period of 1 to 5 days. Among the above inhibitors, only U0126 (10 micromol/L) completely blocked the effects of FGF9 on Runx2 gene promoter activity.</p><p><b>CONCLUSIONS</b>Our data showed that FGF9 can affect Runx2 gene promoter activity in MC3T3-E1 and C2C12 cells. The action of FGF9 appears to depend partly on the mitogen-activated protein kinase kinase/mitogen-activated protein kinase pathways in C2C12 cells.</p>

RET gene cys 634 trp mutation in a multiple endocrine neoplasia type 2A kindred / 中国医学科学院学报

<p><b>OBJECTIVE</b>To identify the genotype of RET gene in one multiple endocrine neoplasia type 2A (MEN2A) kindred.</p><p><b>METHODS</b>Genome DNA was extracted from peripheral blood leucocytes. The DNA sequence of gel-purified polymerase chain reaction (PCR) products was determined with the previously reported 6 pairs of primers of PCR amplification of 10, 11, 13, 14, 15, and 16 exons of RETgene.</p><p><b>RESULTS</b>No abnormalities were found in exon 10, 13, 14, 15, and 16. C to G replacement in nucleotide 14 996 of exon 11 was identified in DNA samples obtained from both peripheral blood of 2 affected brothers. This missense point mutation arisen in heterozygosity and caused a substitution of Cys to Trp residue at codon 634 ( Cys 634 Trp) in RET protein.</p><p><b>CONCLUSION</b>The genotype of the family is identified as Cys 634 Trp substitution of RET gene.</p>

Effects of different human parathyroid hormone 1-34 administration on SaoS-2 cells / 中国医学科学院学报

<p><b>OBJECTIVE</b>To observe the effects of different human parathyroid hormone 1-34 (hPTH1-34) administration on SaoS-2 cells, and explore the mechanism of bone formation improvement.</p><p><b>METHODS</b>Each cycle covered 48 h. SaoS-2 cells were continuously or intermittently stimulated by 50 ng/ml hPTH1-34 for 1, 3, 6, 12, and 24 h in each cycle. Total RNA was extracted by Trizol kit. Alkaline phosphatase (ALP), osteocalcin or bone Gla-containing protein (BGP) and cyclic adenosine monophosphate (cAMP) levels were measured by chemical method, radioimmunoassay and competitive protein binding method, respectively. c-fos gene expression was semi-quantified by reverse transcription polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>ALP level was time-dependently increased in 1, 3 and 6 h stimulation, especially in 3 and 6 h (compared with control, P < 0.01; P < 0.05 or P < 0.01 compared with continuous stimulation). The cAMP level was time-dependently increased in 3 and 6 h incubation (P < 0.05 compared with control and continuous stimulation). Intermittent hPTH1-34 stimulation had more effects on cAMP level than continous action (P < 0.001). hPTH1-34 intermittent stimulation of 1, 3, and 6 h enhanced c-fos gene expression time-dependently.</p><p><b>CONCLUSIONS</b>Intermittent hPTH1-34 stimulation has a stronger effect on osteoblast than continuous action, especially in 3, 6 h in each cycle intermittent stimulation. The synchronous responses of c-fos, ALP and cAMP to hPTH1-34 suggest that hPTH1-34 affect Saos-2 cells through cAMP dependent protein kinase A (PKA) pathway and c-fos gene paly an important role.</p>

Changes of urinary deoxypyridinoline crosslink/creatinine in rats after ovariectomy and anti-osteoporotic intervention / 中国医学科学院学报

<p><b>OBJECTIVE</b>To observe the changes of urinary deoxypyridinoline crosslink/creatinine (UDpd/Cr) in rats after OVX and intervention by estrogen and bisphosphonate and investigate the possible application of deoxypyridinoline in osteoporosis diagnosis and treatment.</p><p><b>METHODS</b>40 female 6-month-old virginal Wistar rats were divided into 5 groups, ovariectomized or sham ovariectomized. (1) Ovxb (n = 8): sacrificed at 6 weeks after OVX; (2) Sham (n = 8): sham ovariectomized; (3) Ovxe (n = 8): sacrificed at 14 weeks after OVX; (4) O + E (n = 9):OVX + 17 beta estradiol [20 micrograms/(kg.d) ih]; (5) O + C (n = 7):OVX + cimadronate [0.2 mg/(kg.d)]; Treatment started 6 weeks after OVX and lasted 8 weeks. Rats in group 2-5 were sacrificed at 14 weeks after OVX. Urinary and serum biochemical parameters were measured, pQCT scanning of femur, bone biomechanical test in femur were determined.</p><p><b>RESULTS</b>OVX resulted in increasing of UDpd/Cr 133.3% (P < 0.01). The ratio of UCa/Cr also increased in OVX groups but without any significant compared with Sham (P > 0.05). UDpd/Cr were reduced by 54.6% and 51.8% (P < 0.01) in O + E, O + C group respectively compared with Ovxe. The significant negative correlationships were found between UDpd/Cr and bone mass, BMD and biomechanic characteristics.</p><p><b>CONCLUSIONS</b>UDpd/Cr ratio is a sensitive bone resorption marker, a marked changes were observed when the rats ovariectomized or treated with estradiol and cimadronate. There were best correlation between UDpd/Cr and bone mineral density and bone biomechanic characteristics. It is fair to apply UDpd/Cr ratio for osteoporosis diagnosis and treatment.</p>

Association of polymorphisms of vitamin D receptor gene start codon and 3'-end region with bone mineral density in postmenopausal women / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To determine whether vitamin D receptor(VDR) gene start codon polymorphisms and 3'-end region polymorphisms exerted a combined influence on bone mineral density(BMD) in Han postmenopausal women in Beijing area.</p><p><b>METHODS</b>The VDR Fok I and 3'-end region genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism in 110 unrelated postmenopausal women. BMD was measured at the lumbar spine (L(2-4)), femoral neck(Neck), Ward's triangle(Ward's) and trochanter (Troch) using duel-energy X-ray absorptiometry.</p><p><b>RESULTS</b>The frequencies distribution of Fok I, Apa I, Bsm I and Taq I alleles in this cohort all followed the Hardy-Weinberg equilibrium. No significant association of Fok I, Apa I or Taq I genotype with BMD in postmenopausal women was found when these polymorphisms were considered independently, except for Bsm I genotype. When a combined analysis of VDR gene Fok I and 3'-end region polymorphisms was carried out, cross-genotyping Fok I and Apa I polymorphisms was significantly associated with BMD at the L(2-4) (P<0.001), and cross-genotype of Fok I and Taq I was also significantly associated with BMD at the Neck and Troch sites (P<0.05). However, cross-genotyping Fok I and Bsm I polymorphisms was not significantly associated with BMD. Cross-genotyping Apa I and Bsm I or Taq I polymorphisms was not associated with BMD in postmenopausal women, either.</p><p><b>CONCLUSION</b>Although Fok I polymorphisms of VDR gene were not significantly associated with BMD in postmenopausal women, VDR gene Fok I and 3'-region polymorphisms (Apa I and Taq I) had a combined effect on the BMD in postmenopausal women.</p>

Precision of rat BMD measurements by dual-energy X-ray absorptiometry in vitro / 中国医学科学院学报

<p><b>OBJECTIVE</b>To evaluate the precision of rat bone mineral density (BMD) measurements by Norland Excellplus dual-energy X-ray absorptiometry (DXA) and to investigate the BMD changes in ovariectomized (Ovx) rats in vitro.</p><p><b>METHODS</b>(1) The coefficients of variation (CV) for BMD measurements at various skeletal regions were repeatedly determined by DXA in 10 Wistar rats in vitro. (2) BMD in lumbar vertebra (L5) and both sides of femurs was measured in total 90 rats. And (3) changes in BMD between Ovx and sham rats were compared.</p><p><b>RESULTS</b>(1) The short-term CVs of BMD measurements in different regions by DXA were as follows, 1.58% for lumbar vertebra (L5), 0.90% for left femur, and 0.86% for right femur, respectively. The long-term CVs were 2.22% for lumbar vertebra (L5), 1.09% for left femur, and 1.20% for right femur. (2) The BMD values in 90 Wistar rats were (127.5 +/- 12.3) in lumbar vertebra (L5), (82.6 +/- 11.3) in corpus vertebra (L5'), (150.7 +/- 10.6) in left femur and (149.9 +/- 10.6) mg/cm2 in right femur, respectively. The correlation coefficient of BMD measurements between left and right femurs was 0.792 (P < 0.001). (3) In Ovx group, the BMD values of corpus vertebra (L5') and distal femurs were significantly decreased, that was 10.0%-17.5% lower in comparison with sham group (P < 0.001).</p><p><b>CONCLUSIONS</b>Measurement of rat BMD in vitro by Norland Excellplus DXA is a useful method, and it can reflect the changes in rat bone masses with good precision.</p>

Distribution of calcium-sensing receptor gene polymorphism and its association with serum calcium level in patients with primary hyperparathyroidism / 中华内分泌代谢杂志

0.05).Conclusion The distribution of G990R CASR genotype in PHPT patients is different from healthy women,and R allele is higher in PHPT group.Among PHPT patients,A986S and G990R polymorphisms are associated with serum calcium and ICa levels.Patients with S or G allele have lower levels of serum calcium and ICa.A986S genotype is also associated with serum PTH level and patients with S allele have relatively lower level of PTH.