BACKGROUND: Embryonic stem cells can be induced to differentiate into neural precursors under certain conditions, and they can effectively integrate with host cells after transplanted into healthy or injured central nervous system, and then repair and rebuild the injured nerve tissue.OBJECTIVE: To observe the effects of transplantation of neural precursors induced by embryonic stem cells on the recovery of neurological function in mice with spinal cord injury.DESIGN: A randomized controlled animal trial.SETTING: Research Center of Developmental Biology, Shanghai Second Medical University.MATERIALS: Twenty-eight C57/BL6J mice of 6-8 weeks old, both sexes, were used. Mice embryonic stem cell strain S8 and carrier LacZ labeling genes were provided by Shanghai Research Center of Developmental Biology. High-glucose Dulbecco's modified Eagle media (DMEM), β-mercaptoethanol (BME), mice leukemia inhibitory factor (LIF) and mitocin-C were all from GIBCO attachment induction medium, which were provided by Shanghai Research Center of Developmental BiologyMETHODS: The experiment was carried out in the central laboratory of developmental biology of Shanghai Second Medical University from April 2003 to April 2004. The embryonic stem cells were cultured and induced to differentiate into neural precursors by means of attachment induction. The mice were anesthetized and made into models of spinal cord hemisection on the T9-T10 plan. The mice were randomly divided into three groups: sham operated group (n =9): Only T9-T10 spinous process and corresponding lamina of vertebra were removed, then the skin was sutured layer by layer;ransplantation group (n =10): After spinal cord hemisection, embryonic stem cells were injected into the vertebral canal about 1 cm away from the injured site; model group (n =9): DMEM was injected into the region around the injured site.The mice were evaluated with Basso, Beattie, Bresnahan (BBB) locomotor rating scale to observe the recovery of neurological function at 1, 2, 4, 6 and 8 weeks (the score ranged from 1 to 21 points, the higher the scores, the better the recovery of neurological function). At 8 weeks, the survival and differentiation of embryonic stem cells at the injured site of spinal cord were observed using X-Gal staining in each group. The positively stained sections with X-Gal at the injured site of spinal cord were detected with fluorescent immunohistochemical staining.MAIN OUTCOME MEASURES: ① Recovery of neurological function; ② Survival and differentiation of embryonic stem cells at the injured site of spinal cord; ③ Results of fluorescent immunohistochemical staining.RESULTS: ① The BBB scores in the transplantation group at 1, 2 and 4 weeks were higher than those in the model group (P ＜ 0.01). ② Survival and differentiation of embryonic stem cells at the injured site of spinal cord: In the transplantation group, there were X-Gal positively stained cells in the tissue sections of the injured spinal cord of mice,the cytoplasm was blue with nucleoli in it, i.e. the cells derived from embryonic stem cells, which were not observed in the sham-operated group and model group. In the transplantation group, the cells derived from embryonic stem cells, which were implanted to the injured spinal cord, distributed around the injured sites, and integrated with the surrounding tissue and had similar form with the surrounding cells. ③ At the injured site, X-Gal positively stained cells in the transplantation group aiso expressed neurofilaments (the specific marker of neurons), but did not express GFAP.CONCLUSION:The embryonic stem cells were cultured and induced to differentiate into neural progenitors, and they could survive, migrate and differentiate into neurons after transplantation, but there was no obvious improvement of neurological function.

BACKGROUND: Embryonic stem cell (ESC) is a kind of highly undifferentiated totipotent cell. It can proliferate and maintain its totipotency in the system cultured in vitro. It is one of most promising stem cells in thetreatment of central nerve injury.OBJECTIVE: To observe the survival and migration of induced transplanted ESC in mice with spinal injury and hypoxic-ischemic encephalopathy.DESIGN: A completely randomized grouping design, controlled animal experiment.SETTING: Laboratory of Developmental Biology Research Center of Shanghai Second Medical University.MATERIALS: Sixty C57/BL6J mice, of clean grade and either gender, aged 6 to 8 weeks (n =30) and 7 days (n =30)were provided by the Shanghai Experimental Animal Center, Chinese Academy of Sciences [Permission No, SCXK (hu)2003-0003]. This animal experiment was approved by Animal Ethics Committee. Mouse ESC strain S8, labeled LacZ marker gene (Provided by Shanghai Developmental Biology Research Center). X-gal dyeing reagent (Sigma Company).METHODS: This experiment was carried out in the laboratory of Shanghai Developmental Biology Research Center (Shanghai Key Laboratory) from October 2002 to December 2003. ① Experimental grouping of spinal injury: Sixteen C57/BL6J successful mice models, aged 6-8 weeks, were randomized into 2 groups: experimental group (n =8), in which, following right spinal semi-sectioning, derivated cell suspension for inducing the in vitro differentiation of ESC was injected at 1 cm away from injury through vertebral canal, and control group (n =8), in which, following right spinal semi-sectioning, phosphate buffer solution (PBS) was injected at the peripheral region of injury. ② Hypoxic-ischemic encephalopathy experimental grouping: Sixteen successful C57/BL6J mice models, aged 7 days, were randomized into 2 groups: experimental group (n =8), following ligation of right common carotid artery, mice were placed in the closed container containing 0.08 volume fraction of oxygen and 0.92 volume fraction of Nitrogen gas, and taken out 1.5 hours later; 3 μL ESCs were injected into the right cerebral ventricle at about 1 week, and control group (n =8), in which, the same amount of PBS was injected into the right cerebral ventricle. ③ At 12 weeks after transplantation, the survival and migration of induced ESCs labeled by Lac-Z in the spinal cord and brain were observed by zymologic method.MATN OUTCOME MEASURES: Survival and migration of ESCs in the central nervous system.RESULTS: ①After being induced in vitro and transplanted to spinal injured region, ESCs differentiated into neural precursor cells. Neural precursor cells could survive in the injured region and migrate to 5 mm away from injured region.Immunohistochemistry proved that the neural precursor cells of transplanted ESCs could differentiate into neurons.Morphologically, it was proved that neural precursor cells-derived from ESCs could well integrate peripheral tissue. ② The induced ESCs were injected into the lateral cerebral ventricle of mice. Derived ESCs widely distributed in the injured hippocampal region, cerebral cortex ventricle choroid plexus, vascular endothelium and other regions, and integrated peripheral tissue, which were similar to adjacent cells in morphology, suggesting that induced ESCs also could survive for long time and far migrate.CONCLUSION:The induced ESC can survive and migrate in the host injured brain and spinal cord, and the migration of ESCs is more obvious in the brain than in the spinal cord.

Objective To investigate the effects of transplantatation human endothelial progenitor cells onneurological functional recovery from spinal cord injury model in rats,survival of transplanting cells and differentia-tion. Method The human endothelial progenitor cells were provided by Shanghai Developmental Biology Labora-tory.Forty SD rats were made for the animal model of spinal cord complete transection.Thirty survival SD rats wererandomly divided into three groups:sham operation gronp(group A, n = 10),operation/cell group(greup B, n =10) and operation/DMEM group (group C, n = 10).Suspension containing (hEPCs 6×106) was transplanted in-to the vertebral canal around injured spinal cord. In group C, equal volume of DMEM was injected insbead in the same way as in the group B. The BBB score was obtained 2,4,6,8 weeks after injection, Immunohistochemistry andin-situ hybridization were used to observe the cells survival and differentiation in the spinal cord. The BBB test wasperformed to study the functional improvement of cells. The SAS version. 1.3 software for statistics was to studyethology and functional improvement. The sum of ranks was checlced with Kruskal-Wallis Test and Nemenyi test.Results There were statistically significant differences in BBB scoring between group A and group B as well asgroup C after operation (P<0.05). The BBB score in group B was higher than that in group C after 2,4,6 and8 weeks,but lower than in group A. The hybridization in-situ and immunohistochemistry showed that transplantedcells survived for 8 weeks after transplantation and expressed specific characteristics for ancestral cell and differentiated into vascular endothelial cell (VEC). Conclusions After transplantation, hEPCs can survive, differenti-ate into vascular endothelial cell,and improve spinal cord function as compared with control group.

AIM: Some researches show that stem cell transplantation for damaged spinal cord can improve the function of damaged spinal cord. But the studies about bone marrow mononuclear cell transplantation for injuried spinal cord are seldom. We transplanted fresh bone marrow mononuclear cells isolated from rats into rat models of injured spinal cord to explore the effect of bone marrow mononuclear cells for injured spinal cord functions, nerve regeneration, neovascuarization and long-term effect. METHODS: Experiments were performed in the Experiment Center of Developmental Biology of Shanghai Second Medical University from October 2005 to April 2006. The laboratory is Specific-pathogen free grade. ①Female clean SD rats aged 8 weeks weighting 200-220 g were offered by Animal Experimental Centre of Chinese Academy of Sciences. Animal intervention met the animal ethical standard. ②Rat bone marrow mononuclear cells were isolated from the tibia and the femur by Ficoll-Paque density gradient centrifugation. Rat models of spinal injury were established. The 22 successfully established rat models were divided into 2 groups. Rat models in a model plus cell group (n =11) received the complete T9-10 transection of spinal cord, and then bone marrow mononuclear cells were transplanted into the vertebral canal. Rat models in a model plus DMEM group (n =11) received the complete T9-10 transection of spinal cord, and then DMEM was injected into adjacent region. Rat models in a sham operation group (n =9) received T9-10 spinous process and lamina of vertebra incision and then the incision was sutured. ③Hybridization in situ and immunohistochemistry technique were used to determine the survival of implanted cells in host spinal cord. BBB scale system was applied to assess the functional recovery of spinal cord nerves. RESULTS: ①There was no significant difference in postoperative score at each time point in the sham operation group. The score was 0 point in the model plus DMEM group. The function of spinal cord did not recover. The function of spinal cord became better at weeks 2, 4, 6 and 8 in the model plus cell group. There were significant differences as compared with the model plus DMEM group and the sham operation group (P

Objective:To investigate the possible mechanism of mesenchymal stem cells (MSC) secreting hepatocyte growth factor (HGF).Methods:① C57BL/6 mouse mesenchymal stem cells (mMSC) were cultured in vitro, and mMSC with high expression of chemokine receptor 7 (CXCR7) were transduced by lentivirus plasmid. Blank control group and empty carrier control group were set at the same time. After 20 generations of cell culture, the transfection efficiency was identified by fluorescence microscopy and flow cytometry. The mRNA expression levels of CXCR7 in mMSC were detected by real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR). ② mMSC with passage number 4-6 were divided into MSC control group [MSC-blank group, 100 μg/L lipopolysaccharide (LPS) was added to wild-type MSC], highly expressed CXCR7 group (MSC-OE-CXCR7 group, 100 μg/L LPS was added to mMSC transduced by lentivirus plasmid with high expression of CXCR7), highly expressed CXCR7 control group (MSC-OENC-CXCR7 group, 100 μg/L LPS was added to mMSC transduced by no load lentivirus plasmid), CXCR4 inhibitor group (MSC-IE-CXCR4 group, 100 μg/L LPS was added to mMSC after 0.1 mg/L CXCR4 inhibitor TC14012 pretreatment for 24 hours), and CXCR4 inhibitor control group (MSC-IENC-CXCR4 group, 100 μg/L LPS was added to mMSC after DMEM culture medium with equal amount of TC14012 pretreatment for 24 hours). Cells in each group were collected after treatment with LPS, and mRNA expression of inhibitor of differentiation-1 (ID-1) was detected by RT-PCR. The cell supernatant was collected, and the levels of HGF were detected by enzyme linked immunosorbent assay (ELISA). Results:① The high expression of CXCR7 for mMSC which were transduced through lentivirus plasmid were successfully constructed detected by fluorescence microscope and flow cytometry. Compared with the blank control group, the expression of CXCR7 mRNA in the lentivirus with high expression of CXCR7 group was significantly increased (2 -ΔΔCt: 5.81±0.97 vs. 1.02±0.12, P < 0.05). There was no significant difference in CXCR7 mRNA expression between the empty carrier control group and the blank control group (2 -ΔΔCt: 0.95±0.22 vs. 1.02±0.12, P > 0.05). ②Compared with the MSC-blank group, high expression of CXCR7 in MSC-OE-CXCR7 group or inhibition of CXCR4 in MSC-IE-CXCR4 group could induce high expression of ID-1 mRNA in mMSC (2 -ΔΔCt: 5.56±0.66, 2.47±0.58 vs. 1.00±0.10, both P < 0.05) and increase HGF exocrine level (ng/L: 632.02±149.98, 217.21±40.53 vs. 108.53±24.62, both P < 0.05). However, there were no significant differences in ID-1 mRNA expression and HGF exocrine level of mMSC among MSC-OENC-CXCR7 group, MSC-IENC-CXCR4 group and MSC-blank group [ID-1 mRNA (2 -ΔΔCt): 1.01±0.27, 1.21±0.32 vs. 1.00±0.10, HGF (ng/L): 133.56±25.19, 107.11±25.30 vs. 108.53±24.62, both P > 0.05]. Conclusion:High expression of CXCR7 or inhibition of CXCR4 in MSC can increase the expression of ID-1 and promote the secretion of HGF, thus promoting pulmonary microvascular endothelial repair.

OBJECTIVETo explore the value of of CD, MPO, Ki-67, C-MYC positive rates in the pathological tissues and C-MYC gene of patients with T-LBL/ALL for predicting Prognosis.

METHODSNinty cases of T-LBL/ALL patients in our hospital were selected and included in the T-LBL/ALL group, and 30 cases of lymphnode reactive hyperplasia were selected as control group. Immunohistochemical staining was used to detect the changes of CD, MPO, Ki-67 and C-MYC positive rate in 2 groups, and the changes of C-MYC gene were detected by fluorescence in situ hybridization.

RESULTSIn 90 patients with T-LBL/ALL, there were CD1a34 cases (37.8%), CD367 cases (74.4%), epsilon CD347 cases (52.2%), CD785 cases (94.4%), CD1033 cases (36.7%), CD3422 cases (24.4%), CD4348 cases (53.3%), CD45RO46 cases (51.1%), CD9988 cases (97.8%), TDT85 cases (94.4%); and CD23, CD20, and MPO all were negative; Ki-67>80% 47 cases (52.2% cases), Ki-67≤80%, 43 cases (47.8%). In 90 T-LBL/ALL patients, the positive rate of C-MYC (66.7%) was significantly higher than the control group (positive rate 0.0%) (P< 0.05); the Ki-67 index, mediastinal widening of T-LBL/ALL patients and the positive rate of C-MYC positively were correlated (P< 0.05). The overall survival rate (44.0%) of C-MYC negative patients was significantly higher than that of C-MYC positive patients (0.0%). The overall survival rate of C-MYC negative patients was significantly higher than that of C-MYC positive patients (P< 0.05).Ann Arbor staging, LDH, bone marrow involvement, mediastinal widening, Ki-67 positive index, and C-MYC protein expression of patients with T-LBL/ALL did not correlated with increased C-MYC gene breakage and copy number (P> 0.05).

CONCLUSIONThe overall survival rate of C-MYC positive patients decreases, which positively correlates with Ki-67 positive index and mediastinal width, suggesting that the prognosis of the patients with C-MYC protein expression is poorer.

Clostridium tyrobutyricum is suitable for simultaneous saccharification and fermentation of lignocellulosic. It can produce butyric acid, acetic acid as its main fermentation products from a wide variety of carbohydrates such as glucose, xylose, cellobiose and arabinose. In order to decrease acetic acid content and increase butyric acid content in C. tyrobutyricum, we replaced genes on the acetic acid fermentation pathway with genes on the butyric acid fermentation pathway. Three genes were selected. They were acetyl-CoA acetylrtansfers gene (thl) which is the key enzyme gene associated with acetic acid fermentation pathway from Clostridium acetobutylicum, erythromycin gene (em) from plasmid pIMP1 and phosphotransacetylase gene (pta) which is the key enzyme gene associated with butyric acid fermentation pathway from C. tyrobutyricum. We fused these genes with pUC19 to construct nonreplicative integrated plasmids pUC19-EPT. Then we transformed pUC19-EPT into C. tyrobutyricum through electroporation. The recombinant transformants grown on plates containing erythromycin were validated by PCR. A mutant whose pta gene was displaced by thl gene on the chromosome was selected. In the fermentation from glucose, the mutant's yield of butyric acid is 0.47, increased by 34% compared with wild type; and the yield of acetic acid is 0.05, decreased by 29% compared with wild type.
Acetic Acid ; analysis ; metabolism ; Acetyl-CoA C-Acetyltransferase ; genetics ; Butyric Acid ; analysis ; metabolism ; Clostridium tyrobutyricum ; genetics ; metabolism ; Fermentation ; Genetic Engineering ; methods ; Glucose ; metabolism ; Industrial Microbiology ; Lignin ; metabolism ; Mutation ; Phosphate Acetyltransferase ; genetics

OBJECTIVETo systematically observe the clinical effect on chronic obstructive pulmonary disease (COPD) at the stable stage, differentiated as cold phlegm blocking the lung type, treated with acupoint sticking therapy during the dog days and the three nine-day periods after the winter solstice so as to propose the latest clinical idea and theoretic evidence for the treatment of COPD.

METHODSOne hundred and fifty cases of COPD at stable stage, which were in accordance with the inclusive standard were randomly divided into three groups, named group A (treatment in dog days and the three nine-day periods after the winter solstice), group B (treatment in dog days) and group C (treatment in the three nine-day periods after the winter solstice), 50 cases in each group. The ingredients (Semen Brassicae, Euphoribia Kansui, Asarum, Rhizome Corydalis, Cinnamon, ginger juice) and doses of herbal medicine plaster were same in each group. The herbal plaster was applied to Feishu (BL 13), Shenshu (BL 23), Dazhui (GV 14), Tiantu (CV 22), Danzhong (CV 17) and Zhongfu (LU 1). In group B, the treatment was given once on the 1st day of each dog-day period, totally 3 treatments were included. In group C, the treatment was given once on the 1st day of each nine-day periods after the winter solstice, totally, 3 treatments were involved. In group A, the treatment was given once on the 1st day of each dog-day period and each nine-day periods after the winter solstice separately, totally 6 treatments were required. The therapeutic effect was evaluated in 4 aspects, named comprehensive clinical efficacy, survival quality (the scores for symptoms, activity limitation and influence on daily life), the attach frequency and pulmonary function.

RESULTSThe total effective rate was 88.0% (46/50) in group A, which was superior to 76.0% (38/50) in group B and 70.0% (35/50) in group C separately (P < 0.01, P < 0.001). The results of the attack frequency, clinical symptom score and pulmonary function indices after treatment were all improved apparently as compared with those before treatment in each group (all P < 0.01). All the above indices in group A were improved much apparently as compared with the other two groups (P < 0.01, P < 0.001). Except for the level of forced vital capacity (FVC), the results of clinical symptom score and the other pulmonary function indices in group B were all improved significantly as compared with group C (P < 0.05, P < 0.001).

CONCLUSIONAcupoint sticking therapy during different season of the year achieves a superior clinical efficacy for the patients with COPD at stable stage. This therapy can reduce the attack frequency and improve the survival quality and pulmonary function for the patients. It is concluded that the efficacy of the treatment in dog days and the three nine-day periods after the winter solstice is superior to simple dog-day treatment and the treatment in the three nine-day periods after the winter solstice, and the efficacy of dog days treatment is better than that in the three nine-day periods after the winter solstice.

Acupuncture Points ; Adult ; Aged ; Aged, 80 and over ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Humans ; Lung ; metabolism ; physiopathology ; Male ; Middle Aged ; Mucus ; chemistry ; metabolism ; Pulmonary Disease, Chronic Obstructive ; drug therapy ; metabolism ; physiopathology ; Seasons ; Vital Capacity

Objective:The three-dimensional(3D) reconstruction method of calf perforator vessel was explored to provide the basis for clinical safe and accurate harvesting of calf chain perforator flap.Methods:Gelatin-lead oxide is automatically perfused the femoral artery of 3 fresh adult lower limbs specimens. Data were collected after the CT scan of the full limb, 3D reconstruction of bones, arteries and their perforators was processed in Mimics Research 19.0 software. Dynamic display of vascular hierarchy and adjacent relationships by adjusting CT values. And show the anatomical region of each perforator artery in different colors, measure the feeding area of the single perforator using Scion Image software. The 3D printed calf bone and vascular model can be used for direct observation. The specimen anatomy was measured according to a perforator vessel parameter with an outer diameter of 0.5 mm through the deep fascia. The number of perforators, vessle pedicle length, and outer diameter were observed and statistically analyzed.Results:The digitized 3D reconstruction images can clearly show the 3D structured of the calf artery and perforator vessels and micro vessels, as well as the course, distribution characteristics of blood vessels, and the 3D relationship with adjacent structures. The printed 3D model visually observes the hierarchy of the perforator vessels and the anastomosis relationship between the perforators. 3 calf specimen arteries with perforator artery 22±4, vessel pedicle length (46.7±18.3) mm, outer diameter (0.8±0.3) mm, the average blood supply area of the single perforator (38.2±10.7) cm 2. Conclusion:The Mimics Research 19.0 software should be used to dynamically reconstruct the 3D structure of the branch vessel by adjusting the CT value, which improves the display effect of the perforator vessels. The 3D printing model makes the source, course, diameter and distribution range of the perforator vessels visually visible, which provides experimental support for the clinical construction of digital calf chain perforator flap harvest scheme for patients.

Objective:The three-dimensional(3D) reconstruction method of calf perforator vessel was explored to provide the basis for clinical safe and accurate harvesting of calf chain perforator flap.Methods:Gelatin-lead oxide is automatically perfused the femoral artery of 3 fresh adult lower limbs specimens. Data were collected after the CT scan of the full limb, 3D reconstruction of bones, arteries and their perforators was processed in Mimics Research 19.0 software. Dynamic display of vascular hierarchy and adjacent relationships by adjusting CT values. And show the anatomical region of each perforator artery in different colors, measure the feeding area of the single perforator using Scion Image software. The 3D printed calf bone and vascular model can be used for direct observation. The specimen anatomy was measured according to a perforator vessel parameter with an outer diameter of 0.5 mm through the deep fascia. The number of perforators, vessle pedicle length, and outer diameter were observed and statistically analyzed.Results:The digitized 3D reconstruction images can clearly show the 3D structured of the calf artery and perforator vessels and micro vessels, as well as the course, distribution characteristics of blood vessels, and the 3D relationship with adjacent structures. The printed 3D model visually observes the hierarchy of the perforator vessels and the anastomosis relationship between the perforators. 3 calf specimen arteries with perforator artery 22±4, vessel pedicle length (46.7±18.3) mm, outer diameter (0.8±0.3) mm, the average blood supply area of the single perforator (38.2±10.7) cm 2. Conclusion:The Mimics Research 19.0 software should be used to dynamically reconstruct the 3D structure of the branch vessel by adjusting the CT value, which improves the display effect of the perforator vessels. The 3D printing model makes the source, course, diameter and distribution range of the perforator vessels visually visible, which provides experimental support for the clinical construction of digital calf chain perforator flap harvest scheme for patients.