Objective To study the effect of substance P ( SP) on int racellular f ree calcium concent ration in human poorly-differentiated gast ric cancer cell in vitro. Methods Human gast ric cancer cell line MKN45 was cultured in RPMI 1640. Then the cells were loaded with specific calcium fluorescent probe Furu23/ AM. ASN21377642 (NK21 receptor antagonist) , Nicardipine (calcium channel blocker) and different concent rations of SP were used to treat gast ric cancer cells. The concent ration changes of int racellular free calcium were detected by laser scanning confocal microscope. Results It was found that 10 , 50 and 100 nmol/ L SP could significantly increase the int racellular free calcium concent ration of gast ric cancer cells in Hanks solutions , which contain ext racellular calcium ( P lt;0. 05) , and the change was in a dose-dependent manner ( P lt; 0. 05) . When there was ext racellular calcium existed ,the increasing amplitude of intracellular f ree calcium concent ration was significantly higher than that when there was no extracellular calcium ( Plt; 0. 05) . And when Hanks solutions were pretreated with ASN21377642 and Nicardipine , the effects of 100 nmol/ L SP were partly inhibited , and the concent rations of int racellular f ree calcium were significantly lower than those in group s without pret reatment s ( P lt; 0. 05) . Conclusion SP can significantly increase free calcium concent ration in the gastric cancer cells. Releasing of stored calcium in the cells and influx of extracelluar calcium may contribute to the elevation of int racellular free calcium concentration.
【Abstract】Objective To observe the bacteria in cholesterol stones by electronic microscope and to explore the role of bacteria in the stone formation.Methods Twelve patients with cholelithiasis underwent operations (male 6, female 6, average age 54.6 years） with cholecystolithiasis 5, extrahepatic and intrahepatic bile duct stone 1, common bile duct stone combining with gallstone 6. The cholesterol stones were observed by electronic microscope.Results There were bacterial structures in the cholesterol stones and cholesterol crystals.Conclusion There are bacteria in the core and peripheral of cholesterol stones, which suggests that bacteria may play an initial role in the formation of cholesterol stones.
Objective To investigate the assembl ing and cl inical appl ication of the video output system util izing teaching sight glass of surgical microscope. Methods Between June 2009 and April 2010, 10 patients with craniocervical junction malformation were treated by the method of transoral-transpharyngeal approach with the microscope and videooutput system under the direct vision. There were 6 males and 4 females with an average age of 32 years (range, 13-52 years). Three cases had the history of injury and 7 cases had no history of definite injury. The disease duration was from 10 months to 12 years (median, 5 years). The main cl inical symptoms were brevicoll is or torticoll is; 2 patients had malformation appearance and 4 patients had occi put-cervical pain. The physical examination showed that all patients had the symptoms that upper cervical cord was damaged; the imaging examination showed that all patients had basilar invagination, atlantoaxial dislocation, and ossification. Before and after operations, the functions of nerve were evaluated by Japanese Orthopaedic Association (JOA) scoring, the improvement rate was calculated to evaluate the efficacy. Results By the video output system assembly, 15.1 mill ion pixels high-definition images could be collected and reached 1 920 × 1 080 pixels video camera, so assistants or medical students could watch the cl inical operation directly. All patients had no neural injury or cerebrospinal fluid leakage during operation. Basilar invagination and atlantoaxial dislocation were corrected. Infection at incision occurred in 1 patient; other incisions healed by first intention without early compl ication. All patients were followed up 6-16 months (mean, 13.5 months). The average JOA score was increased from 10.2 preoperatively to 15.5 at 6 months postoperatively with an improvement rate of 77.9%. At 12 months after operation, bony fusions were achieved. Conclusion The miscroscope and video output system can improve the effectiveness of the original surgical microscope. It makes visual fields much clearer and operations more accuratewith a few compl ications.
Objective To comprehend the pathological features and possible pathogenesis of avascular necrosis of the femoral head (ANFH) by morphology and immunohistochemical observation of osterix (OSX) and adiponectin through in vitro traumatic and non-traumatic ANFH specimens, so as to provide a theoretical basis for cl inical treatment. Methods Sixty-six ANFH specimens were collected from 66 cl inical cases undergoing hip replacement surgery. Twenty-four cases of traumatic ANFH (group A) included 17 males and 7 females, aged 21 to 70 years with an average of 56.5 years; 23 cases of steroid-induced ANFH (group B) included 16 males and 7 females, aged 56 to 72 years with an average of 61 years; and 19 cases of alcohol ic ANFH (group C) were males, aged 55 to 67 years with an average of 58.5 years. Bone tissue was got from weight-bearing and non-weight-bearing area of the femoral head respectively. The basic pathological changes was observed by HE staining under the optical microscope, and the percentage of empty bone lacuna and the percentage of trabecular bone area were calculated. The morphological changes of ANFH in different groups were observedby scanning electron microscope (SEM). OSX and adiponectin expression were detected by immunohistochemical technique. Results Gross of the femoral head surface in each group was rough, collapse, articular cartilage loss, osteophyte formation; cross section: dark red in group A, and yellow in groups B and C. HE staining showed that weight-bearing area of ANFH have similar morphological features in three groups. In non-weight-bearing area of groups B and C, the fat cells in bone marrow markedly increased and were hypertrophic; however there were more fibrous tissue in group A. There were statistically significant differences (P lt; 0.001) in the percentage of empty bone lacuna of the weight-bearing and non-weight-bearing area among three groups. There were no statistically significant differences (P gt; 0.05) in the percentage of trabecular bone area among three groups. The SEM observation showed that three groups had similar pathological changes. Brown granules for OSX and adiponectin positive substance were mainly located in the osteoblast of bone marrow of the femoral head. There was statistically significant difference (P lt; 0.05) in the average absorbency (A) value of OSX between group A and groups B, C, but there was no statistically significant difference (P gt; 0.05) between groups B and C. While there was no statistically significant difference (P gt; 0.05) in the A value of adiponectin among three groups. Conclusion Hormones and alcohol necrosis have more obviously fatty degeneration, but the repair capacity of traumatic femoral head necrosis is ber than that of hormones and alcohol necrosis. Alcohol and hormones have inhibitory action on the OSX-mediated osteogenic differentiation. Hormones and alcohol may not affect osteoblast expressing adiponectin and its receptors.
Objective The combined appl ication of green fluorescent protein (GFP) and confocal laser scanning microscope three-dimensional reconstruction (CLSM-3DR) were used to monitor the construction and in vivo transplantation of tissue engineered bone (TEB), to provide for technology in selection of scaffolds and three-dimensional constructional methods. Methods After bone marrow mesenchymal stem cells (BMSCs) were isolated from a 2-year-old green goat by a combination method of density gradient centrifugation and adherent culture, and the expressions of CD29, CD60L, CD45, and CD44 in BMSCs were detected by flow cytometry. Plasmid of pLEGFP-N1 was ampl ified, digested by enzymes (Hind III, BamH I, Sal I, and Bgl II), and identified. Transfection of pLEGFP-N1 into PT67 cells was performed under the help of l iposome. Positive PT67 cells were picked out with G418, and prol iferated for harvesting virus. Based on the titre of virus, after BMSCs were infected by virus containing pLEGFP-N1, GFP positive BMSCs were collected and prol iferated for seeding cells. TEB was fabricated by GFP positive BMSCs and decalcified bone matrix (DBM) and observed by CLSM-3DR for the evaluation of the distribution and prol iferation of seeding cells. After TEB was transplanted in the defect of goat femur, CLSM was used for observing the survival and distribution of GFP positive cells in the grafts. Results The isolated cells were fibroblast-l ike morphous, with the positive expression of CD29 and CD44, and negative expression of CD60L and CD45. The digested production of pLEGFP-N1 was collected for ionophoresis, whose results showed the correct fragment length (6 900 bp). The virus of pLEGFP-N1 was harvested by transfection of pLEGFP-N1 into PT67 cells and used for further infection to obtain GFP positive BMSCs. The prol iferated GFP positive BMSCs and DBM were used for fabrication of TEB. The distribution, prol iferation, and migration of BMSCs in TEB were observed by CLSM-3DR. GFP positive cells also were observed in images of TEB graft in goat femur 28 days after transplantation. Conclusion The BMSCs labeled by GFP in three-dimensional scaffold in vivo were monitored well by CLSM-3DR. It suggests a wide use potency in monitoring of three-dimensional cultured TEB.
Objective To study the influence of the immersed time by hydrogen dioxide on the characteristics of bovine cancellous bone granules in various periods. Methods Ten 24-month-old Qinchuan bovine, male or female, weighing 150-170 g, were selected. Cancellous bone granules from metaphysic of bovine long bone were cut into cubes of 5 mm × 5 mm ×5 mm and immersed by 8.8 mol/L hydrogen dioxide for 0, 12, 24, 36, 48, 60 and 72 hours separately. Determination of ash, scanning electron microscope, X-ray energy spectrum and micro CT were used to investigate the changes of composition, structure and qual ity of bone. Results With the immersed time increasing, the contents of organics in the bone cancellous were reduced gradually, and obviously decreased during the periods of 0 to 24 hours and 60 to 72 hours (P lt; 0.05). The contents of calcium and phosphorus decreased gradually, they could not be detected almost after 60 days (P lt; 0.05). Bone mineral density and bone mineral content were decreased obviously after 60 hours (P lt; 0.05). The bone trabecula became sl immer and trabecular spacing became larger. Conclusion Hydrogen dioxide can be used to remove the antigen in xenogeneic bone; however as the time increasing (more than 60 hours) the composition and structure will be damaged. Thus it is important to control the immersed time for maintaining the biological characteristics of xenogeneic bone substitute as well as el iminating antigen by hydrogen dioxide.
Objective To investigate the feature and regularity of the collagen change in bone healing during bone lengthening. Methods Bone lengthening model was made in the middle segment of the rabbit tibia. Five days after the model was established, the bone was lengthened 1.5 mm perday for 14 days. The rabbits were put to death after elongation, 7,14,21,30,40,50,60 and 70 days after elongation. The distracted area of the bone was imbedded with paraffin. After being stained by the picric acidsirius red staining, the slice was observed under polarized microscope. Results The features of the collagen change in the distracted bone were as follows: ① In the fibrous tissue of the distracted area during lengthening period and the early stage after lengthening, there was not only collagen Ⅲ but alsomuch collagen Ⅰ. ② Collagen Ⅰ, Ⅱ and Ⅲ were observed in the cartilage. ③ Collagen Ⅰ, Ⅱ and Ⅲ were also observed in the pseudogrowth plate. ④ Collagen Ⅰ took the dominance during lengtheningperiod and the late stage after lengthening. Conclusion New bone formation in bone lengthening is under the distracted force, so the collagen changes have different features compared with that in fracture healing. Collagen Ⅰ, Ⅱ and Ⅲcan be identified by picric-acid-sirius red staining and polarized microscope, so a new method for studying the collagen typing in bone repairing is provided.
OBJECTIVE: To investigate the expression and distribution of platelet derived growth factor receptor-beta(PDGFR-beta) in normal skin and keloid and to discuss its biological function in keloid formation. METHODS: 1. To detect the expression and distribution of PDGFR-beta in normal skin and keloid tissue by immunohistochemistry; 2. To detect the receptor expression in vitro by Flow cytometry (FCM); 3. To detect the subcellular distribution of receptor by Laser confocal microscope. RESULTS: 1. Immunohistochemistry showed that normal skin and keloid tissue were almost the same in expression but different in distribution of PDGFR-beta; 2. There was more expression of PDGFR-beta in normal fibroblasts than that in keloid fibroblasts in vitro by FCM; 3. Laser confocal microscope revealed that the PDGFR-beta concentrated on the surface of cell membrane in keloid fibroblasts, but in normal skin fibroblasts, the receptors were coagulated on the nuclear membrane and intranucleus. CONCLUSION: Compared with the fibroblasts in vivo, there was a difference of the PDGFR-beta expression in fibroblasts in vitro, more expression of PDGFR-beta in normal fibroblast than that in keloid fibroblast in vitro; and the subcellular distribution of PDGFR-beta was different in normal skin and keloid fibroblasts. The characteristics of the expression and distribution of PDGFR-beta in keloid may contribute to the formation of keloid.
In order to get high-resolution glomerulus image with large field of view (FOV), stitching multiple small FOV images with high-resolution is necessary. Directly stitching images without properly correction is not acceptable and cannot afford any significant assistance in pathological diagnosis for intensity inhomogeneity and geometric distortion. Therefore we proposed a method of distortion correction and intensity inhomogeneity correction of glomerulus transmission electron microscope (TEM) image. In this paper, we firstly describe how these two distortions degrade images. Secondly, based on the TEM imaging system, image acquisition model and distortion correction model were proposed. Then according to these two models, distortions were greatly degraded and stitching results were improved by respectively applying two corrections, intensity inhomogeneity correction and geometric distortion correction. With the method proposed here, the result was improved significantly and stripes, fuzzy and artifacts were decreased dramatically. Our method has been proved to be valid to solve the problems of TEM glomerulus image distortion and at the same time to improve the result of multiple TEM glomerulus image stitching.
ObjectiveTo investigate the effectiveness of posterior microscopic mini-open technique (MOT) decompression in patients with severe spinal canal stenosis resulting from thoracolumbar burst fractures.MethodsThe clinical data of 28 patients with severe spinal canal stenosis caused by thoracolumbar burst fractures, who were treated by posterior microscopic MOT, which performed unilateral or bilateral laminectomy, poking reduction, intervertebral bone graft via spinal canal, and percutaneous pedicle screw fixation between January 2014 and January 2016 were retrospectively analyzed. There were 21 males and 7 females with a mean age of 42.1 years (range, 16-61 years). The involved segments included T11 in 1 case, T12 in 4 cases, L1 in 14 cases, and L2 in 9 cases. According to AO classification, there were 19 cases of type A3, 9 of type A4. According to American Spinal Injury Association (ASIA) grading, 12 cases were grade C, 13 grade D, and 3 grade E. The time between injury and operation was 3-7 days (mean, 3.6 days). To evaluate effectiveness, the changes in the visual analogue scale (VAS), percentage of anterior height of injured vertebrae, Cobb angle, rate of spinal compromise (RSC), and ASIA grading were analyzed.ResultsAll patients were performed procedures successfully. The operation time was 135-323 minutes (mean, 216.4 minutes). The intraoperative blood loss was 80-800 mL (mean, 197.7 mL). The hospitalization time was 10-25 days (mean, 12.5 days). The incisions healed primarily, without wound infection, cerebrospinal fluid leakage, or other early complications. All the 28 patients were followed up 12-24 months (mean, 16.5 months). No breakage or loosening of internal fixation occurred. All fractures healed, and the healing time was 3-12 months (mean, 6.5 months). Compared with preoperative ones, the percentage of anterior height of injured vertebrae, Cobb angle, and RSC at immediate after operation and at last follow-up and the VAS scores at 1 day after operation and at last-follow were significantly improved (P<0.05). There was no significant difference in the percentage of anterior height of injured vertebrae and Cobb angle between at immediate after operation and at last follow-up (P>0.05). But the RSC at immediate after operation and VSA score at 1 day after operation were significantly improved when compared with those at last follow-up (P<0.05). The ASIA grading at last follow-up was 1 case of grade C, 14 grade D, and 13 grade E, which was significantly improved when compared with preoperative ones (Z=3.860, P=0.000).ConclusionMOT is an effective and minimal invasive treatment for thoracolumbar AO type A3 and A4 burst fractures with severe spinal canal stenosis, and it is beneficial to early rehabilitation for patients.