Objective To evaluate the character of the collagen-chitosan-chondroitin sulfate scaffold seeded with rat adipose tissue-derived stromal cells. Methods A dipose tissue were harvested from 6 weeks old Wistar rats and the stromal cells were harvested by type Ⅰ collagenase and then cultured in vitro. Type Ⅰ collagen was fully mixed with chitosan, freeze-dried and cross-linked with chondroitin sulfate, then freeze-dried again and sterilized by ethylene oxide. The pore diameter, water content, porosity of the scaffold were tested. The adipose tissue-derived stromal cells were digested, seeded into the plates, scaffold, and cen-trifuged into pellet, and then induced into cartilage. MTT detection for cell proliferation was done. After 3 weeks, the cell morphology, and cell proliferation and adhesion were observed, and chondrngenic differenti-ation was also analyzed. Results The pore diameter, water content, porosity tested for the scaffold showed an appropriate form. Cell proliferation showed faster in the scaffold and pellet culture system after 5 day, there was still cell proliferation in the scaffold system after 14 days but no obvious changes in the pellet cul-ture system; ceils on the scaffold proliferated densely showed by histological staining, but there was a scaf-fold structure residues in the inner layer. The finding of type Ⅱ immunohistochemistry stain showed that cells express strong positive for type Ⅱ collagen in the scaffold and pellet culture system whereas it was weakly positive in the plate culture system; the specific mRNA for cartilage, type Ⅱ collagen, aggrecan and SOX-9 were expressed in all three systems showed by RT-PCR, but type X collagen was expressed continu-ously in the plate culture system and expressed after 21 days in the pellet culture system, whereas it was not detected in the collagen-chitosan-chondroitin sulfate scaffold system. Conclusion The parameters of the collagen-chitosan-chondroitin sulfate scaffold were suitable in our study. The results suggested that it can promote the adipose tissue-derived stromal cells proliferation and chondrogenic differentiation better than the plate and pellet culture systems and maintain the phenotype of chondrocytes well; it is the optimal choice for cartilage tissue engineering in the future.

BACKGROUND: The implanted cartilage calls can synthesize cartilage matrix as cartilage in cartilage tissue enginsedng, and the density of implanted cells is the key point.OBJECTIVE: To evaluate the effect of cell seeding concentration on the chondrogenic differentiation of the adipose dadved sromal cells (ADSCs).DESIGN, TIME AND SETTING: The in vitro cellular-scaffold observation was performed at the cytobiological laboratory of China Medical University from November 2007 to July 2008.MATERIALS: Six male SD rata with clean grade were supplied by the Experimental Animal Center of China Medical University.METHODS: Totally 5 g/L type ; collagen solution and 20 g/L chitosan was mixed in a mould with volume ratio of 7:3, after lyophillization, it was cut into pieces with 5 mm ～ 5 mm x 2 mm, followed by crosslinking with ethanol contained of 2% chondroitic acid at room temperature. After washing with double distilled water and freeze drying, the chitosan-collagen-chondroitin sulfate copolymar matrices scaffolds were harvested. ADSCs isolated from rat inguinal fat pads were digested with collagenase and trypsase. The prepared scaffolds were randomly divided into 3 groups, and the third passage cells with density of 2×10 9/L,2×10 109/L, and 2×10 11/L were seeded into chitosan-coflagen-chondroitin sulfate scaffolds, and cultured in chondrogenic medium for 3 weeks.MAIN OUTCOME MEASURES: The expression of cartilage specificity gene was detected by hematoxylin-eosin staining, type Ⅱ collagen immunohistochemical staining and RT-PCR.RESULTS: Hematoxylin-eosin staining showed that after 3 weeks of culture, the cell proliferated and differentiated well, especially in 2x101～/L group, more extrocelluer matrices were produced and cartilage lacuna-structure could be seen. The type Ⅱ collagen was positive expressed in each group, which showed a gradually increasing tendency with the cell seeding concentration increasing. RT-PCR showed that the expression of proteoglycen and type Ⅱ collagen mRNA were slowly increased. However the expression of Ⅹ collagen mRNA was decreased with increasing cell seeding concentration.CONCLUSION: The chitosan-collagen-chondroitin sulfate copolymer matrices can provide an appropdate environment for the generation of cartilage-like tissues and high call seeding concentration of 2×1010/L facilitate ADSCs to differentiate into cartilage.

Objective To investigate the usefulness of the helical CT in demonstrating the abnormal blood vessels of intralobar pulmonary sequestration. Methods In fourteen patients with suspicion of pulmonary sequestration, contrast enhanced helical CT was performed. Clinical presentation included hemoptysis (2), pulmonary infection (8), and asymptomatic (4). Fourteen cases underwent helical CT examination with different scanner and scan parameters. Five CT angiography (CTA) studies were performed by single-section helical CT scanner (Hispeed Advantage, GE, USA) with 3 mm slide thickness, 4.5 mm/s table speed and 1.5 mm reconstruction index. Three CTA studies were done by multisection CT scanner (MX8000 four-slice CT, Marconi, USA) with a collimation of 2.5 mm, resulting in an effective slice width of 3.2 mm. Non-ionic contrast media (iodine 300 mg I/ml) was administered at a rate of 2.5 ml/s via the antecubital vein. The optimal opacification of the upper abdominal aorta were achieved through the use of the Smart Prep (Hispeed Advantage) program or by using the Boluspro ultra (MX8000). Three-dimensional reconstruction was performed with the workstation. Five cases were scanned by using single-section helical CT scanner with 5 mm collimated section and 5 mm/s table speed. And in one patient CT scan was performed with 10 mm section thickness and 10 mm/s table speed. Results CT findings were pulmonary mass (11) and well-defined nodules (3). The lesions were located in right lower lobe (2) and left lower lobe (12). Helical CT identified the aberrant arterial supply in all cases. The origin of the anomalous artery was the descending thoracic aorta (2), upper abdominal aorta (11) and splenic artery (1). These findings were confirmed by operation. Conclusion The combination of axial and 3D images in helical CT, especially the multislice helical CT, is helpful in the assessment of the abnormal blood vessels of intralobar pulmonary sequestration. It can substitute to the angiography in the diagnosis of pulmonary sequestration.

The effects of oxygen partial pressure on cryopreservation of the cells with organ preservation solution were explored. Hypoxic UW solution was made by purging the UW solution with argon. The pig proximal tubule epithelial cells (LLC-PK1 cells) were cryopreserved in hypoxic UW solution (Ar-UW group) or standard UW solution (UW group) at 4 degrees C for 48 h. Trypan blue staining and LDH detection were performed to evaluate the injury of the cells. The results showed that the oxygen partial pressure in Ar-UW group was significantly declined from 242+/-6 mmHg to 83+/-10 mmHg. After cryopreservation at 4 degrees C for 48 h, LDH leakage rate and Trypan blue-stained rate in Ar-UW group were (11.3+/-3.4)% and (10.5+/-4.7)%, respectively, which were significantly lower than in UW group [(49.5+/-6.9)% and (47.6+/-9.3)% respectively, both P<0.01]. It was concluded that lower oxygen partial pressure of UW solution was more beneficial to the cryopreservation of LLC.
Adenosine ; Allopurinol ; Cell Hypoxia ; Cell Line ; Cryopreservation ; Cryoprotective Agents ; Epithelial Cells/*cytology ; Glutathione ; Insulin ; Kidney Tubules, Proximal/cytology ; Organ Preservation Solutions ; Oxygen/pharmacology ; Raffinose ; Swine ; Tissue Preservation/methods

In order to explore the method to prepare hypoxia UW solution and the stability and preservation of hypoxia UW solution, UW solution was purged by argon or air for 15 min or 60 at a flow rate of 0.8 or 2 L/min, and the oxygen partial pressure of UW solution was detected. The hypoxia UW solution was exposed to the air or sealed up to preserve by using different methods, and the changes of oxygen partial pressure was tested. The results showed that oxygen partial presure of 50 mL UW solution, purged by argon for 15 min at a flow rate of 2 L/min, was declined from 242+/-6 mmHg to 83+/-10 mmHg. After exposure to the air, oxygen partial pressure of hypoxia UW solution was gradually increased to 160+/-7 mmHg at 48 h. After sealed up by the centrifuge tube and plastic bad filled with argon, oxygen partial pressure of hypoxia UW solution was stable, about 88+/-13 mmHg at 72 h. It was concluded that oxygen of UW solution could be purged by argon efficiently. Sealed up by the centrifuge tube and plastic bag filled with argon, oxygen partial pressure of UW solution could be stabilized.
Adenosine/chemical synthesis ; Allopurinol/chemical synthesis ; Anoxia ; Glutathione/chemical synthesis ; Insulin/chemical synthesis ; Organ Preservation/*methods ; Organ Preservation Solutions/*chemical synthesis ; Oxygen/*analysis ; Partial Pressure ; Raffinose/chemical synthesis

Objective To investigate the expression and significance of Bmi-1 in pancreatic cancer.Methods The expression of Bmi-1 mRNA was detected in pancreatic cancer and matched adjacent normal tissues by using real-time PCR in 22 cases of human pancreatic carcinoma.Bmi-1 and p16 protein were determined in 61 cases of human pancreatic carcinoma with immunohistochemistry and their relationship with clinicopathologic parameters was analyzed.Results The expression of Bmi-1 mRNA was 0.314 ±0.040 in cancerous tissues, and 0.143 ±0.056 in adjacent normal tissues, and the difference was statistically significant (P ＜0.01 ).36 cases (59.0%) of PC were detected with positive expression of Bmi-1 protein, and 34 cases (55.7%) with no expression of p16 protein.The expression of Bmi-1 was significantly correlated with lymph node metastasis ( P = 0.020 ) and tumor differentiation ( P = 0.018 ); the expression of Bmi-1 was negatively correlated with the expression of p16 ( P = 0.033 ).Conclusions Expression of Bmi-1 was increased in pancreatic cancer, which may be involved in the malignant behavior of pancreatic cancer by inhibiting p16 expression.

Objective To investigate the effect of dexamethasone on the postoperative cognitive function in rats.Methods One hundred and eighty Sprague-Dawley rats,aged 18-20 months,weighing 400-600 g,were randomly allocated into 3 groups (n =60 each)∶ control group (C group),surgery group (S group) and dexamethasone group (D group).In groups S and D,the rats were anesthetized with 5％ chloral hydrate 4-6 ml/kg and underwent abdominal surgery.The rats in group D received intraperitoneal injection of dexamethasone 10 mg/kg at the beginning of anesthesia,while the rats in group C underwent no surgery and received intraperitoneal injection of normal saline 1 ml/kg instead.Six rats in each group were chosen at 3 h and 7 days after surgery and sacrificed,and their brains were immediately removed for detection of the expression of OX42 (a specific marker for activation of microglia) in hippocampus.Another 6 rats in each group were chosen at 3 h,and 1,3 and 7 days after surgery and sacrificed,and their brains were immediately removed for detection of the expression of IL-1β mRNA and TNF-α mRNA in hippocampus.Cognitive function was assessed by Morris water maze test and fear conditioning test.Results Compared with group C,the escape latency was prolonged,the frequency of crossing the original platform was decreased,the postoperative freezing time was shortened,and the expression of OX42 after surgery and IL-1β mRNA and TNF-α mRNA at 3 h and 1 and 3 days after surgery was up-regulated in groups S and D (P ＜ 0.05 or 0.01).Compared with group S,the escape latency was shortened,the frequency of crossing the original platform was increased,the postoperative freezing time was prolonged,and the expression of OX42 at 3 h after surgery and IL-1β mRNA and TNF-α mRNA at 3 h and 1 and 3 days after surgery was down-regulated in group D (P ＜ 0.05 or 0.01).Conclusion Dexamethasone can inhibit the over-activation of microglia and reduce the inflammatory response,thus improving cognitive function in rats.

Objective To evaluate the effect of surgical trauma on the cognitive function and expression of hepcidin and ferroportin 1 (FP1) in hippocampus in aged rats.Methods One hundred male Sprague-Dawley rats,aged 18 months,weighing 400-500 g,were randomly divided into 2 groups with 50 rats in each group:control group (group C) and surgical trauma group (group ST).The rats were anesthetized with chloral hydrate,but underwent no operation in group C.The rats Were anesthetized with chloral hydrate and underwent 30 min of modified exploratory laparotomy in group ST.Ten rats were chosen from each group at 24 h after operation and the cognitive function was assessed using Morris water-maze test for 6 consecutive days.Ten rats were sacrificed on 1st,3rd,5th and 7th days after beginning of Morris water-maze test and brains were removed for determination of hepcidin and FP1 expression in hippocampus by PCR and Western blot.Results Compared with group C,the escape latency was significantly prolonged,the time of staying at the original platform quadrant and frequency of crossing the original platform were decreased on 3rd,4th and 5th days after beginning of Morris water-maze test,and the expression of hepcidin was up-regulated and the expression of FP1 was down-regulated at each time point in group ST (P ＜ 0.05).Conclusion Surgical trauma can decrease the cognitive function in aged rats and the mechanism may be related to up-regulation of hepcidin expression and down-regulation of FP1 expression in hippocampus.

Objective To investigate the effect of surgical trauma on the cognitive function and activation of microglias in hippocampus in rats of different ages.Methods Seventy-two male Sprague-Dawley rats,aged 3-4months,were randomly allocated into 2 groups:adult control group (n =30) and adult surgery group (n =42).Seventy-two male Sprague-Dawley rats,aged 18-20 months,were randomly allocated into 2 groups:aged control group (n =30) and aged surgery group (n =42).The rats were anesthetized with 5% chloral hydrate 4-6 ml/kg and underwent exploratory laparotomy in surgery groups,while normal saline 1 ml/kg was injected intraperitoneally in control groups.Morris water maze test was performed at 1-7 days after surgery.Fear conditioning test was performed 1 day after surgery to evaluate the space and fear memory abilities.The animals were sacrificed on 1st,3rd and 7th days after surgery and hippocampi were removed for measurement of OX42 expression in microglias by immunohistochemistry.Results Compared with adult control group,the percentage of freezing time in total time was significantly decreased,and OX42 expression in microglias was up-regulated on 1st day after surgery (P ＜ 0.05),and no significant change was found in the escape latency and the number of crossing the original platform in adult surgery group (P ＞ 0.05).Compared with aged control group,the escape latency was significantly prolonged,the number of crossing the original platform was decreased,the percentage of freezing time in total time was decreased,and OX42 expression in microglias was up-regulated on 1st and 3rd days after surgery in aged surgery group (P ＜0.05).Conclusion Surgical trauma decreases fear memory ability,but exerts no effect on the space memory ability in adult rats.Surgical trauma decreases the space and fear memory abilities in aged rats,which maybe related to activation of microglias in hippocampus.

Objective To explore the clinical effect of autogenous bone,allograft bone and BMP synthetic bone in the treatment of lum-bar spondylolisthesis,and provide more basis to choose transplant material.Methods A total of 96 patients with lumbar spondylolisthesis were chosen as research subjects,who were cured in our hospital from January 2014 to January 2015.They were divided into group A(who were treated with autogenous bone),group B(who were treated with allograft bone)and group C(who were treated with BMP synthetic bone), according to prospective study method.The indicators of the operation,postoperative adverse reactions,change of intervertebral disc height and bone graft fusion rate of three groups were compared.Results Difference of the operation indexes of three groups had no statistical sig-nificance(P >0.05).The incidences of adverse reactions in group A and group C had no statistically significant difference(P >0.05),but both less than that in group B,with statistically significant difference(P <0.05).The intervertebral disc height after 6,9,12 and 18 months in group A and group C had no statistically significant difference(P >0.05),but both more than that in group B,with statistically significant difference(P <0.05).Bone graft fusion rate of group C was faster than that of group A and goup B,and the graft fusion rate in 18 month in group A and C had no statistically significant difference(P >0.05).And the fusion rates of group A and C in each period were significantly higher than that of group B,with statistically significant difference(P <0.05).Total effective rate of neurologic improvement in group A and group C had no statistically significant difference(P >0.05),but both better than that of group B,with statistically significant difference (P <0.05).Conclusion BMP synthetic bone used in lumbar spondylolisthesis has the same clinical effect as autologous bone.But BMP synthetic bone has faster bone graft fusion rate than autologous bone.And it is beneficial to patients’recovery.