ObjectiveTo evaluate the efficacy of XiaochengqiMixture (XM) on promoting healing of colonic stoma. MethodsForty Wistar rats were divided into two groups randomly after colonectomy: experimental group (n=20) and control group (n=20). In early postoperatively stage rats were given gastric administration of XM in the experimental group and pure water in the control group. On day 3, 7, and 14 after establishment of animal models, laparotomy was performed in two groups of rats, respectively. Anastomotic stoma and surrounding tissues were harvested to detect the context of hydroxyproline and collagen fiber proportion by Masson dying. ResultsOn day 3 after establishment of animal models, hyperplastic collagen with small fiber was observed while no fasciculus was found. Hydroxyproline context and collagen fiber proportion of rats were higher in experimental group than those in control group (Plt;0.05). On day 7 after operation, many fasciculuses were found in two groups of rats, hydroxyproline context and collagen fiber proportion of rats were higher in experimental group than those in control group (Plt;0.01). On day 14 after operation, fasciculuses became bigger and more regular in arrangement, but there was no significant difference between the two groups (Pgt;0.05). ConclusionXM is capable of promoting healing of colonic stoma and might prevent the occurrence of anastomotic fistula.
Objective To establish a purified model of rat retinal ganglion cells （RGCs） cultured by serum-free medium，and provide a good cell model to investigate the damage of RGCs in glaucoma，retinal ischemia，and degenerative retinopathy. Methods Two monoclonal antibodies，anti-rat SIRP（OX-41）against rat macrophage and antibody against rat Thy-1（OX-7），were used to purify and characterize RGCs from 1-3-day old Sprague-Dawley（SD）rats by means of two-step filtration.Purified RGCs were cultured in serum-free neurobasal medium containing B27 and ciliary neurotrophic factor（CNTF） meeting the neuronal cellrsquo;s special requirements.Photomicrographs illustration，immunfluorescence staining of Thy-1，calcein-acetoxymethyl ester（calcein-AM）fluorescence images were used to observe and identify cultured retinal cells and purified RGCs. Results Among the primary cultured rat retinal cells，91% were retinal neurons.Protuberances of RGCs were seen after cultured for 24 hours.At the4th to 8th day，many cells had uniform configuration，large body，and long protuberances. At the 14th day，over 60% cells maintained viability.Immunoflurescence staining of Thy-1 showed the purity of RGCs was about 90%. The results of calcein-AM staining，which stained the living cells only，showed large cell body of RGCs and most of RGCs had a protuberance whose length was twice longer than the diameter of the cells. Conclusion RGCs cultured by serum-free medium has uniform size，good configuration，and high purity，which is adapt to the research of damage of RGCs caused by various factors and to evaluate the protective effects of neuroprotective agents. (Chin J Ocul Fundus Dis, 2006, 22: 200-203)
Objective To explore a rapid histological preparation method to observe morphology and composition distribution of tendon collagen fascicle and endotendinum. Methods Taking porcine superflexor tendon of foot as an example, tendons were sliced into sections with 6 μm by frozen section technology, after which general observation of the section integrity was carried out. After fixed with 10% neutral buffered formalin and performed with HE staining, the tissue integrity and ice crystal formation were observed under microscope. Sections were then divided into 5 groups by different methods of dyeing. Group A: Priodic acid-Shiff (PAS) staining; group B: Masson staining; group C: reticular fibers staining; group D: immunohistochemical and immunofluorescent staining of type Ⅲ collagen; group E: the sections were baked at 65℃ for 10 minutes and stained with Masson. The composition distribution of tendon collagen fascicle and endotendinum in different groups were observed. Results From general observation, the frozen section of tendon tissue was complete and continuous. Although the tissue integrity in the tendon sections could be seen and no ice crystal was formed, the composition distribution could not be identified by HE staining. The entire tendons in groups A, B, and C were dyed, and the composition distribution of collagen fascicle and endotendinum could not be identified. The endotendinum in group D was stained weakly positive for type Ⅲ collagen alone, and the two components were differentiated dyed but the contrast was not obvious. In group E, the collagen fascicle and endotendinium were differentiated dyed and the two components in tendon tissue were clearly visible. Conclusion The morphology and the composition distribution of tendon collagen fascicle and endotendinum can be characterized rapidly and accurately, using a combination of baking at 65℃ for 10 minutes and Masson staining after porcine superflexor tendons were sliced by frozen section technology.
Objective It is reported that transforming growth factor β1 (TGF-β1) has the protective effects on the articular cartilage in osteoarthritis (OA). To investigate the significance of the expressions of matrix metalloproteinase 9 (MMP-9), TGF-β1 mRNA and corresponding proteins in OA. Methods The specimens of articular cartilage and synovium were collected from voluntary donators, including 60 cases of OA (experimental group) and 20 cases of traumatic amputation,cruciate l igament rupture, discoid cartilage injury, and menisci injury (normal control group). The pathological changes were observed by HE staining. MMP-9 and TGF-β1 protein expressions were detected by immunohistochemical technique, and the mRNA expressions of MMP-9 and TGF-β1 were detected through in situ hybridization technique; and their correlation was analysed. Results HE staining showed: shrinkage, necrosis, and irregular arrange of the articular chondrocytes, extracellular matrix fracture, hypertrophy and hyperplasia synovium, infiltration of lymphoid and mononuclear cells and prol iferation of many small blood vessels in the experimental group; regular arrangement of the articular chondrocytes, the homogeneously staining matrix, and synovial tissue without chronic inflammation and significant prol iferation in the normal control group. The mRNA and protein expressions of MMP-9 and TGF-β1 were positive in 2 groups. The positive-stained cells included chondrocytes, synovial l ining cells, and vascular endothel ial cells, fibroblasts, and inflammatory infiltrated cells in subsynovial layer. The expressions of mRNA and corresponding protein of MMP-9 and TGF-β1 in the experimental group were significantly higher than those in the normal control group (P lt; 0.01). There was a positive correlation between MMP-9 mRNA and protein expression (r=0.924, P=0.000), and between TGF-β1 mRNA and protein expression (r=0.941, P=0.000) in the experimental group. There was a negative correlation between the expression of MMP-9 protein and TGF-β1 protein (r= — 0.762, P=0.000), and between the expression of MMP-9 mRNA and TGF-β1 mRNA (r= — 0.681, P=0.000) in the experimental group. Conclusion The higher expression of TGF-β1 can protect articular cartilage by down-regulating the expression of MMP-9 of chondrocytes and synoviocytes in OA, which may delay the biological behavior of OA such as occurrence and progress, etc.
Cytogenetic study of 18 colorectal carcinomas confirmed the extensive heterogeneity and the complexity of the karyotypic picture in this tumor.Karyotypic analysis showed that chromosomes 7 and 3 were of the highest chromosomal gaining frequencies(72%,66%) and chromosomal losses were shown in chromosome 17(50%),chromosome5(44%) and chromosome 18(33%).The structual rearrangements frequently involved were 17p(78%),5q(61%),6q,7q,8p,12q,2p,etc.A great number of marker chromosomes and polyploid chromosomes had bad prognosis relatively.According to these results,we conclude that chromosomes 17,5,and 18 may play an important role in the evolution of colorectal cancer.
Objective To reveal the significance of D2-40/CK19 dual immunohistochemistry for micrometastasis of peripancreatic neural plexus in patients with pancreatic cancer. Methods Between January 2006 and January 2007, 44 patients with pancreatic duct adenocarcinoma underwent extended radical resection. Conventional hematoxylin/eosin staining and double immunohistochemical staining using CK19 and D2-40 were used to determine peripancreatic neural invasion and lymphatic vessel invasion (LVI) in peripancreatic neural plexus tissues. Results D2-40 immunohistochemistry showed brown-yellow tube-like lymph vessels. The lymph vessel of peripancreatic nerve plexus followed vascular and perineurium, and the lymph vessel adjacent to peripheral nerve fascicles owned tube-like structure. CK19 immunohistochemistry showed cytoplasm of pancreatic cancer cell was red. The LVI was observed in lymphatic capillaries. Peripancreatic neural plexus invasion was found in 30 cases (68.2%), tumor cell invading presented in lymph vessels of peripancreatic neural plexus in 21 patients (47.7%) with pancreatic cancer. The peripancreatic neural plexus invasion was associated with LVI (P=0.003). The plexus of pancreatic capitalis and celiac plexus were respectively confirmed to be the spot with the highest lymphatic vessel density and the maximal incidence of neural plexus invasion simultaneously. Conclusions Patients with pancreatic cancer should be given the opportunity of radical operation combining related peripancreatic neural plexus as far as possible. The dual immunohistochemical staining with anti-CK19 and anti-D2-40 monoclonal antibodies should be a new method in research of perineural invasion of pancreatic cancer, exhibiting both the pancreatic cancer cells and lymph vessels clearly and distinctly.
In recent years, the application of the long read single-molecule sequencing technology in clinical genetics testing has been increasingly widespread. Due to its sequencing process not requiring amplification and its characteristic of single-molecule sequencing and long reads, it has unique advantages compared to traditional molecular genetics testing such as Sanger sequencing, next-generation sequencing and chromosomal microarray. In this article, its application in the detection of chromosomal structural abnormalities, monogenic diseases, and preimplantation genetic testing for monogenic diseases were discussed. Along with a comparative analysis of the advantages and disadvantages of the long read single-molecule sequencing technology in these areas compared to traditional molecular genetics testing techniques. This article preliminarily explores the application prospects and value of the long read single-molecule sequencing technology in clinical genetics testing.
Objective To investigate the difference of minichromosome maintenance protein 2 (MCM2) mRNA expression among colonic normal mucosa, colonic adenoma and carcinoma. Methods The expressions of MCM2 mRNA were determined by real-time RT-PCR in 12 colonic normal mucosa, 33 colonic adenomas and 12 colonic carcinomas. Data were evaluated by relative expression software tool (REST-XL). Results The expressions of MCM2 mRNA elevated in turn by colonic normal mucosa, adenoma and carcinoma. The expression of MCM2 mRNA in colonic carcinomas was significantly higher than that in adenomas， as well as in normal mucosa (P=0.001), while the difference of MCM2 mRNA expressions between adenoma and normal mucosa was insignificant (P=0.184). Conclusion The difference of MCM2 mRNA expressions between adenoma and carcinoma indicated the potential value on early diagnosis for colonic carcinoma.