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Autologous bone marrow stem cell transplantation for thromboangiitis obliterans：5-year follow-up

Chinese Journal of Tissue Engineering Research.2015;(23):3692-3697. doi:10.3969/j.issn.2095-4344.2015.23.015

BACKGROUND:The assessment for long-term efficacy of chronic ischemic disease is more important than the short-term efficacy assessment, which associates with patient’s long-term quality of life and long-term survival rate. OBJECTIVE:To observe the 5-year folow-up outcomes of autologous bone marrow stem cel transplantation for the treatment of thromboangitis obliterans. METHODS:This study enroled 43 patients of thromboangitis obliterans who underwent autologous bone marrow stem cel transplantation from August 2007 to January 2010 in the Department of Thyroid Vascular Surgery, the First Affiliated Hospital of Xinjiang Medical University. At 1, 2, 3, 4 and 5 years after transplantation, pain, cold sensation, and intermittent claudication distance were folowed up by telephone; changes in limb ulcers were observed. At 1 year after transplantation, venous oxygen partial pressure and oxygen saturation of limbs were reviewed. RESULTS AND CONCLUSION:A total of 38 thromboangitis obliterans patients with complete folow-up data were included in the final analysis. Compared to the preoperation, pain, cold sensation, and intermittent claudication significantly improved. The difference was statisticaly significant (Z values:-4.277,-5.086,-3.574, P < 0.001). Compared with 1-5 years after operation, pain and cold sensation had no statisticaly difference (P >0.05). Intermittent claudication distance had increased. Differences in terms of intermittent claudication distance was statisticaly significant (Z=43.898,P < 0.001). Significant differences in venous oxygen partial pressure and oxygen saturation were detected between preoperation and 1-year posttransplantation (tvalues: 36.790, 43.964,P values: 0.040, 0.037). Above results suggest that autologous bone marrow stem cel transplantation for thromboangitis obliterans obtained stable long-term outcomes.

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Human telomerase reverse transcriptase gene-modified umbilical cord mesenchymal stem cell transplantation for acute kidney injury

Tiansheng WANG ; Jianjun ZHANG

Chinese Journal of Tissue Engineering Research.2015;(23):3686-3691. doi:10.3969/j.issn.2095-4344.2015.23.014

BACKGROUND:The human telomerase reverse transcriptase (hTERT) is one of preferred growth factors for regulating proliferation and directional differentiation, has multiple biological effects, and laids the foundation for geneticaly engineered immortalized stem cel lines. OBJECTIVE: To investigate the effect ofhTERT gene-modified umbilical cord mesenchymal stem cel transplantation on acute kidney injury induced by ischemia and reperfusion in rats. METHODS:The human umbilical cord mesenchymal stem cels were cultured in vitro. Rat models of acute kidney injury induced by ischemia and reperfusion were established. Rat models were randomly divided into three groups. Rats in the control group were injected with 1 mL L-DMEM medium through caudal vein. Rats in the negative transfection group were injected with 1 mL umbilical cord mesenchymal stem cel suspension after empty virus transfection through caudal vein. Rats in the hTERT transfection group were injected with 1 mL umbilical cord mesenchymal stem cel suspension after PLXSN-hTERT transfection through caudal vein. RESULTS AND CONCLUSION:At 3 and 28 days after transplantation, hematoxylin-eosin staining showed renal tubular damage score in the hTERT transfection group < negative transfection group < control group (P < 0.05). At 28 days after transplantation, the number of CM-Dil-positive cels in the hTERT transfection group > negative transfection group > control group (P < 0.05). At 1, 3, 14, and 28 days, serum creatinine and urea nitrogen levels in the hTERT transfection group < negative transfection group < control group (P < 0.05). The results confirm that hTERT gene-modified umbilical cord mesenchymal stem cel transplantation has a significant repair effect on acute kidney injury in rats.

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MicroRNA differential expression in liver cirrhosis rats undergoing human umbilical cord mesenchymal stem cell transplantation

Xiangzhong LIU ; Zhiqiang ZOU ; Guiqiang WANG ; Dong LI ; Zhiying SHAO

Chinese Journal of Tissue Engineering Research.2015;(23):3674-3680. doi:10.3969/j.issn.2095-4344.2015.23.012

BACKGROUND:Human umbilical cord mesenchymal stem cels (hUC-MSCs) can obviously relieve liver cirrhosis, and thereby repair liver injury. However, the molecular mechanism of hUC-MSCs therapy for liver cirrhosis is limited at present, and especialy the non-coding RNA regulation of hepatic gene changes has not been detailed. OBJECTIVE:To investigate the changes of microRNA after hUC-MSCs therapy in rats with liver cirrhosis. METHODS:Liver cirrhosis models were established in rats using carbon tetrachloride subcutaneous injection plus oral administration of alcohol. At 8 weeks after modeling, hUC-MSCs were injectedvia the tail vein once a week for 4 consecutive weeks. At 1 week after the last injection, rat liver tissues were colected for paraffin embedding. Liver RNA was extracted for gene chip analysis. Blood samples were colected and analyzed using an automatic biochemical analyzer to detect the changes of liver function. RESULTS AND CONCLUSION:Alanine aminotransferase, aspartate aminotransferase and gamma-glutamyl transpeptidase were improved significantly after hUC-MSCs therapy. Fat lesions and necrosis of hepatocytes were significantly reduced. MicroRNA expression microarray hybridization analysis and PCR results showed that rno-miR-369-5p, rno-miR-3584-5p and rno-miR-153* were down-regulated during modeling and increased after hUC-MSCs therapy. And rno-miR-93, rno-miR-199a-3p, rno-miR-195, rno-let-7a and rno-miR-19a were firstly up-regulated in the process of modeling and then down-regulated obviously after hUC-MSCs therapy. These results suggest that hUC-MSCs may reverse liver cirrhosis and liver cel damage through up-regulation of rno-miR-369-5p, rno-miR-3584-5p and rno-miR-153*, and down-regulation of rno-miR-93, rno-miR-199a-3p, rno-miR-195, rno-let-7a and rno-miR-19a.

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Effects of cancer stem cell surface marker CD44 in gastric cancer invasion and lymph node metastasis

Shiting LI ; Wencheng TAO

Chinese Journal of Tissue Engineering Research.2015;(23):3669-3673. doi:10.3969/j.issn.2095-4344.2015.23.011

BACKGROUND:Tumor stem cels not only initiate tumorigenesis, but also are involved in the invasion and metastasis of tumor cels. For tumor stem cels, to identify the specific cel surface marker has become a research hotspot. OBJECTIVE:To explore the clinical significance of cancer stem cel surface marker CD44 in gastric cancer invasion and lymph node metastasis. METHODS: CD44 protein expression in specimens of gastric cancer tissue was detected by the immunohistochemical SABC method. The relationship between CD44 protein expression and biological characteristics and prognosis of gastric cancer was detected using Pearsonχ2 test and Cox regression analysis. RESULTS AND CONCLUSION:Among 100 cases of gastric carcinoma, 59 cases (59%) were positive for CD44 protein expression. CD44 protein expression in normal gastric mucosa at above 5 cm from the edge of primary gastric cancer was negative. CD44 protein was widely expressed in tissues of gastric cancer, mainly expressed in the cel membrane, and a smal amount of expression in the cytoplasm. CD44 protein expression in gastric cancer tissue was not correlated with sex of the patients or age (P > 0.05), but was associated with tumor staging and lymph duct tissue infiltration, histological grade, and tumor size (P < 0.05). Deep tumor invasion, high histological grade, big diameter of tumor, and lymph node metastasis could lead to high positive CD44 protein expression. Positive expression of CD44 is an independent prognostic factor affecting postoperative survival (P < 0.05). The results show that the cancer stem cel surface markers CD44 in gastric carcinoma tissues is strongly associated with invasion of gastric carcinoma and lymph node metastasis. High expression of CD44 presents poor prognosis.

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Effect ofp53 inhibitor on viability of human bone marrow mesenchymal stem cells in late-phase amplification

Zebin HE ; Yunhe ZHAO ; Guijiao YANG ; Li LU

Chinese Journal of Tissue Engineering Research.2015;(23):3616-3620. doi:10.3969/j.issn.2095-4344.2015.23.002

BACKGROUND:It is not fuly understood that whetherp53 inhibitor can directly intervene in the viability of bone marrow mesenchymal stem cels and the possible mechanism. OBJECTIVE:To investigate the effect of p53 inhibitor, PFT-α, on the aging process of bone marrow mesenchymal stem cels in late-phase amplification and to discover the key target to delay the replicative senescence of human bone marrow mesenchymal stem cels. METHODS:The expression levels ofp53,p21, andp15 mRNA in human bone marrow mesenchymal stem cels in both early and late-phase amplification were detected by quantitative PCR assay. Then, human bone marrow mesenchymal stem cels in late-phase amplification were respectively treated with 20 μmol/L PFT-α or an equivalent amount of dimethyl sulfoxide for 2 weeks. The positive rate of aging cels was determined by SA-β-Gal staining. The apoptosis was detected by TUNEL staining. Human bone marrow mesenchymal stem cels were treated with 300 μmol/L H2O2 for 30 minutes, and then celular anti-oxidative stress capacity was detected by cel counting kit-8 assay. RESULTS AND CONCLUSION:The quantitative PCR assay showed that the mRNA expression level ofp15, p21 andp53 in human bone marrow mesenchymal stem cels in late-phase amplification was significantly increased (1.45±0.23), (1.51±0.14) and (1.78±0.14) times as much as that in early phase amplification (P < 0.05). The positive rate of aging cels in PFT-α group was significantly lower than that in the dimethyl sulfoxide group[(41±5)%vs. (63±7)%,P < 0.05)]. However, there was no significant difference in apoptosis rate between PFT-α group and dimethyl sulfoxide group. After treatment with H2O2, the absorbance value in the PFT-α group was(1.27±0.13) times as much as that in the dimethyl sulfoxide group (P < 0.001). The above results demonstrate that the activation ofp53 signaling pathway may be an important factor of causing aging of human bone marrow mesenchymal stem cels. Application ofp53 inhibitor PFT-αcan enhance the anti-oxidative stress capacity of human bone marrow mesenchymal stem cels in late phrase amplification.

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Effects of magnetic fields on the differentiation of bone marrow mesenchymal stem cells

Jing ZHANG ; Qi ZHAN ; Yan HUANG

Chinese Journal of Tissue Engineering Research.2015;(23):3744-3749. doi:10.3969/j.issn.2095-4344.2015.23.023

BACKGROUND:Bone marrow mesenchymal stem cels have the ability to differentiate into a variety of non-hematopoietic tissue cels. Effects of magnetic fields on the differentiation of bone marrow mesenchymal stem cels have attracted a lot of attention in recent years. OBJECTIVE:To summarize the effects of magnetic fields on the differentiation of bone marrow mesenchymal stem cels towards osteoblasts, chondrocytes, adipocytes, nerve cels and cardiomyocytes, which provide references for the research and application of tissue engineering seed cels as wel as the clinical applications of magnetic fields. METHODS:The first author performed a data retrieval of PubMed and CNKI databases from 2000 to 2015 to search the articles addressing the effects of magnetic fields on the differentiation of bone marrow mesenchymal stem cels, and reviewed the literatures systematicaly. Finaly, 40 articles were chosen for further analysis. RESULTS AND CONCLUSION:Magnetic fields can promote the differentiation of bone marrow mesenchymal stem cels towards osteoblasts, chondrocytes, nerve cels and cardiomyocytes, and inhibit the differentiation of bone marrow mesenchymal stem cels towards adipocytes. There are optimal frequency and intensity in the induction of magnetic fields on the differentiation of bone marrow mesenchymal stem cels. In general, low-intensity and low-frequency magnetic fields have more obvious effects on bone marrow mesenchymal stem cels. The facilitation of magnetic fields on the differentiation of bone marrow mesenchymal stem cels is also a time-dependent behavior.

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Biocompatibility of porcine bone marrow mesenchymal stem cells-derived bile duct endothelial cells with electrospun nanofibers

Yang YANG ; Jiahua ZHOU ; Xueyan YIN ; Yong XU ; Yang CAO ; Qian XU

Chinese Journal of Tissue Engineering Research.2015;(23):3736-3743. doi:10.3969/j.issn.2095-4344.2015.23.022

BACKGROUND:Repair of extrahepatic biliary tract injury is a difficult problem in the abdominal surgery. Tissue-engineered extrahepatic biliary tract is an ideal selection for this problem. Construction of tissue-engineered extrahepatic biliary tract with excelent performance is a key to related studies. OBJECTIVE:To investigate the biocompatibility of bile duct endothelial cels differentiated by porcine bone marrow mesenchymal stem cels with electrospun nanofibers. METHODS:Porcine bone marrow mesenchymal stem cels were induced toward biliary tract endothelial cels, which were then identified by morphology and RT-PCR. Polylactic-co-glycolic acid (PLGA) nanofiber membranes were prepared by electrospinning. The morphology was determined by scanning electron microscopy and the short-term (2-week)in vitro degradation rate was determined. Adhesion and proliferation of biliary tract endothelial cels on the nanofiber surface was analyzed by calculating the cel adhesion rate and MTT assay, respectively. Cel growth, morphology and distribution on the material surface were observed by fluorescence staining and scanning electron microscopy, respectively. RESULTS AND CONCLUSION: After 4 weeks of directed differentiation of bone marrow mesenchymal stem celsin vitro, cels showed typical morphology of dendritic bile duct endothelial cels and had the expression of CK19. Scanning electron micrographs showed that electrospun materials were continuous nanofibers with diameters between 200 and 500 nm. No significant degradation of the PLGA nanofibers was observed within 2 weeks. Based on the measured cel adhesion rate, MTT assay, fluorescence staining, and scanning electron microscopy, the differentiated cels possessed a good proliferative capacity on PLGA nanofibers. Bone marrow mesenchymal stem cels differentiated into bile duct endothelial cels in vitro. Materials prepared by the electrospinning method had a nanofiber structure, which did not significantly degrade within 2 weeks. Differentiated cels exhibit good biocompatibility with the nanofibers.

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Effects of umbilical cord Wharton’s jelly mesenchymal stem cell transplantation on the expression of inflammatory factors in rats with spinal cord injury

Shanshan MA ; Ruina QU ; Yi TIAN ; Ning YAO ; Yuanbo CUI ; Kang HAN ; Qu XING ; Bo YANG ; Fangxia GUAN

Chinese Journal of Tissue Engineering Research.2015;(23):3729-3735. doi:10.3969/j.issn.2095-4344.2015.23.021

BACKGROUND:The production and release of a large amount of inflammatory factors caused by immune system inflammatory response mainly contributes to secondary spinal cord injury. OBJECTIVE:To investigate the effects of umbilical cord Wharton’s jely mesenchymal stem cel transplantation on repair of injured neurological function and expression of inflammatory factors monocyte chemoattractant protein 1 and interleukin 10 in rats with acute spinal cord injury. METHODS: Eighty-one healthy adult male Sprague-Dawley rats were randomly and equaly divided into sham operation, model and cel transplantation groups, with 27 rats per group. Rats in the latter two groups were subjected to hemisection of the spinal cord to establish acute spinal cord injury models. Rat models in the cel transplantation group received umbilical cord Wharton’s jely mesenchymal stem cel injection (1×106)via the tail vein. Rat neurological function was evaluated using the BBB score at different time points after spinal cord injury. The expression of monocyte chemoattractant protein 1 and interleukin 10 in injured spinal cord tissue was detected using ELISA assay at different time points after spinal cord injury. Migration and neuronal differentiation of umbilical cord Wharton’s jely mesenchymal stem cels in the injured spinal cord tissue were determined using immunohistochemical staining method. RESULTS AND CONCLUSION:Compared with the sham operation and model groups, rat neurological function was significantly recovered in the cel transplantation group (P < 0.05). Compared to the model group, monocyte chemoattractant protein 1 level in the serum and monocyte chemoattractant protein 1 mRNA and protein expression in the injured spinal cord tissue were significantly lower (P < 0.05), but interleukin 10 mRNA and protein expression in the injured spinal cord tissue was significantly higher (P < 0.05), in the cel transplantation group. In the cel transplantation group, umbilical cord Wharton’s jely mesenchymal stem cels could migrate to the injured region and express glial fibrilary acidic protein. These findings suggest that umbilical cord Wharton’s jely mesenchymal stem cels promote rat neurological function recovery by regulating the inflammatory response in the injured spinal cord tissue, which is likely to be one of mechanisms by which transplantation of umbilical cord Wharton’s jely mesenchymal stem cels treats spinal cord injury.

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Effect of platelet lysate on the biological characteristics of human mesenchymal stem cells

Di WU ; Xiaoyun WU ; Yan WU

Chinese Journal of Tissue Engineering Research.2015;(23):3723-3728. doi:10.3969/j.issn.2095-4344.2015.23.020

BACKGROUND:Platelet lysate has been known as a kind of lysate of autologous or alogeneic platelet-rich products. It not only removes the residual cel structure, reduces immunogenicity, but also retains many growth factors. Platelet lysate has been suggested as a substitute for fetal bovine serum to expand mesenchymal stem cels in vitro. OBJECTIVE:To observe the effects of platelet lysate on biological characteristics of human bone marrow mesenchymal stem cels and adipose mesenchymal stem cels, and provide some experimental data for clinical cel therapy and regenerative medicine. METHODS:Platelet lysate was prepared by repeated freezing and thawing from fresh blood. Healthy adult bone marrow and adipose tissue were colected. Human bone marrow mesenchymal stem cels and adipose mesenchymal stem cels were obtained by density gradient centrifugation and type I colagenase digestion. We tested the morphology, cel phenotype, differentiation characteristics, proliferation capacity, colony forming ability and the level of cytokine secretion of bone marrow mesenchymal stem cels and adipose mesenchymal stem cels after cultured with platelet lysis. RESULTS AND CONCLUSION:Bone marrow mesenchymal stem cels and adipose mesenchymal stem cels were successfuly cultured in vitro using platelet lysate. There were no significant differences in morphology, cel phenotype, colony forming ability and the level of cytokine secretion, and chondrogenic, osteogenic and adipogenic capacities between bone marrow mesenchymal stem cels and adipose mesenchymal stem cels. Adipose mesenchymal stem cels had a high cumulative population doublings than bone marrow mesenchymal stem cels (P < 0.05). These findings suggest adipose mesenchymal stem cels had a stronger proliferative ability, and are more suitable for large-scale expansionin vitro cultivation system of platelet lysate compared with bone marrow mesenchymal stem cels.

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Effect of enamel matrix derivatives on the differentiation and proliferation of human periodontal ligament stem cells

Shuang WANG ; Peixun FENG ; Yue CHEN ; Haijuan ZHANG ; Sha LI ; Qinghong BAO ; Limin GUAN

Chinese Journal of Tissue Engineering Research.2015;(23):3716-3722. doi:10.3969/j.issn.2095-4344.2015.23.019

BACKGROUND:The enamel matrix derivative has been used in the clinical treatment of severe periodontitis; however, the mechanism(s) by which enamel matrix derivative promotes periodontal regeneration is stil obscure. OBJECTIVE:To explore the effects of enamel matrix derivatives on the differentiation and proliferation of periodontal ligament stem cels. METHODS:Periodontal ligament stem cels were isolated and identified from human teeth. Cloning forming efficiency, surface antigen expression and pluripotency were detected and identified. Enamel matrix derivatives with different concentrations (20, 50, 100 mg/L) were used to culture periodontal ligament stem cels for 2 and 4 weeks. Colagen synthesis and mineralized nodule formation were detected using Trichrom staining and Von Kosa’s staining, respectively; real-time RT-PCR was employed to detect expressions of colagen type I, osteocalcin, and RUX2; MTT and cel growth rate assay were used to detect the proliferation of periodontal ligament stem cels. RESULTS AND CONCLUSION:Periodontal ligament stem cels were spindle-shaped and showed a higher colony forming efficiency than periodontal ligament cels. The expressions of surface antigens of periodontal ligament stem cels-CD105, CD29, CD45, CD44 were respectively 99.8%, 99.7%, 1.26%, 98.8%, indicating periodontal ligament stem cels have the multilineage differentiation potential. Enamel matrix derivatives improve the colagen synthesis and mineralization nodule formation of periodontal ligament stem cels in a time-dose dependent manner. They also can improve the expression of osteogensis-related genes colagen type I, osteocalcin, RUX2 and proliferation of periodontal ligament stem cels.

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