Objective To observe preserving effect on myocytes in porcine aortic valve replacement with minimal extracorporeal circulation (MECC). Methods 7 pigs were collected as experimental animals and undertook aortic valve replacement with MECC. Morphological and immunofluorescence intensity changes of right atrial and left ventricular tissues were observed. Results HE staining showed that there were not significant changes and edema or injury of myocytes of right atriums and left ventricles between preoperation and postoperation. Immunofluorescence staining showed complement C3b/c in right atrial myocardial tissues after the operation were a little ber, and innate antibody IgG were a little ber in left ventricular myocardial tissues but similarly weak in right atrial myocardial tissues pre- and post-operation. There was not significant changes in HSPG staining in pre-and post-operative right atrial myocardial tissues, but HSPG were obviously weaker in left ventricular myocardial tissues after the operation. Conclusion MECC is effective on support of porcine aorta valve replacement.
Objective To investigate the method of constructing a tissue engineered epidermis with human epidermal cells and polycarbonate membrane, and to establ ish a tissue engineered epidermis with barrier function which is intended to be the replacing model in vitro of skin irritation test. Methods The tissue engineered epidermis was constructed by using polycarbonate membrane as scaffold and stratified differentiated epidermis derived from human keratinocytes. The tissue engineered epidermis was cultured on an inert polycarbonate filter at the air-liquid interface. After 13 days of culture, the composition and structure of tissue engineered epidermis were observed by HE staining, immunofluorescence staining of keratin 10 (K10) amp; K13, K14, laminin,involucrin, and filaggrin, and transmission electronic microscope. The half maximal inhibitory concentration of a substance (IC50) of SDS was determined in the penetration test of tissue engineered epidermis cultured in the absence (control group) or the presence (experimental group) of l i pid supplement for 18 hours. Results The constructed epidermis was similar to normalepidermis, which was consisted of a proliferating basal layer, differentiated spinous layer, granular layer, and stratum corneum. The IC50 values of tissue engineered epidermis cultured in the control group and experimental group were 0.072% (2.36 mmol/L) and 0.183% (6.00 mmol/L), respectively. Conclusion The tissue engineered epidermis constructed on polycarbonate membrane has normal composition and structure and barrier function corresponding to the normal epidermis.
Objective Using chemically extracted acellular methods to treat extracranial section of the canine whole facial nerve, to evaluated its effects on nerve structure and the removal extent of Schwann cells and myel in. Methods Twenty whole facial nerves were exposed from 10 canines [weighing (18 ± 3) kg]. The extracranial trunk of canine facial nerve and its branches (temporal branch, zygomatic branch, buccal branch, marginal mandibular branch, and cervical branch) were dissected under l ight microscope. Twenty facial nerves were divided into the experimental group (n=12) and control group (n=8) randomly. In experimental group, the nerve was extracted with the 3%TritonX-100 and 4% sodium deoxycholate. In control group, the nerve was not extracted. HE staining and immunofluorescence histological stainings for Hoechst33258, P75, Zero, and Laminin were performed. Results After histological staining, it was found that myel in and Schwann cells were removed from the facial nerve while the basal lamina tube remained intact. The whole canine facial nerves (one nerve trunk and multiple nerve branches) had the similar result. Conclusion The canine whole facial nerve has natural structure (one nerve trunk and multiple nerve branches) by extracted with chemically extracted acellular methods, so it is an available graft for repairing the defect of the whole facial nerve.
ObjectiveTo observe the detection of circulating tumor cells (CTCs) in peripheral blood of patients with gastric cancer, and to investigate the relationship between the CTCs and clinicalpathological features of gastric cancer. MethodsSixty cases of gastric cancer from September 2011 to September 2013 of our hospital were as the research object, and compared with 40 cases of benign gastric disease over the same period. These patients' venous blood were collected, the CTCs counts were determined by using the CellTracks AutoPrep fluorescence scaning system, and the relationship between CTCs and clinicopathological features of gastric cancer was analyzed. ResultsThe detection rate of CTCs in gastric cancer patients was 70.0% (42/60), in control group was 7.5% (3/40). The positive rate of CTCs in peripheral blood of patients with gastric cancer was significantly higher than that of benign gastric disease (P<0.05). The positive rate of CTCs in peripheral blood were no correlated with gender, age, N staging, distant metastasis, tumor size, and vascular invasion (P>0.05), but were correlated with the TNM staging of tumor and degree of differentiation (P<0.05). The cumulative survival rates of 12 months and 18 months after operation in CTCs negtive patients with gastric cancer were higher than that CTCs positive patients (P<0.05). ConclusionsThe detection of CTCs is easy to manage and repeatable. The positive rate of CTCs in gastric patients is higher, which can reflect the progression of tumor and serve as the prognostic index in gastric cance patients.
ObjectiveTo introduce a technique of frozen sections for undecalcified bone and discuss its feasibility by observing the fluorescence distribution of the bone and cartilage. MethodsThe male Sprague Dawley transgenic rats at the age of 8 weeks, which express green fluorescent protein were selected to isolate the whole knee sectioned by the undecalcified bone frozen section technique. Under the fluorescence and light microscopy, the fluorescence and structure were observed within the organization of slice. Immunohistochemical staining (collagen type Ⅰ and Ⅱ), HE staining, toluidine blue staining, and Alizarin red staining were performed to observe the distribution of fluorescent substance and cartilage and bone structure. ResultsThe thickness of sections prepared by this technology was 6 μm. General observation showed that the structure of sectioned joint was complete. Under the light microscope, the morphology of cartilage cells, the arrangement of subchondral bone, and trabecular bone traveling could be clearly distinguished. Under fluorescence microscope, green fluorescence was shown in the joint soft tissue, cartilage tissue, and bone tissue; collagen type Ⅰ expressed in the bone tissue, collagen type Ⅱ in cartilage tissue. HE staining and toluidine blue staining could clearly distinguish the morphology of the cartilage layer. Alizarin red staining showed the structural integrity of subchondral bone plate and the organization within the meniscus, and proximal tibia cortical bone continuity. ConclusionThe fluorescence distribution can be directly observe in the bone and cartilage by sectioning of frozen undecalcified bone. This new technology can shorten the cycle of preparing sections.
ObjectiveTo establish a better immunofluorescence protocol to detect co-localization of p53 and mitochondria which may benefit studies aiming to detect mitochondrial expression of proteins.MethodsHeLa cells were treated with hypoxia and the expression of p53 was detected by immunoblotting. HeLa cells were fixed with methanol, methanol: acetone (1: 1, v/v) mixture, and 4% paraformaldehyde, respectively; the former two groups were not permeable, while the latter was penetrated with 0.1% Triton-X 100 and stained with p53 and mitochondria at the same time. After HeLa cells were fixed with 4% paraformaldehyde, the concentration of Triton-X 100 was reduced to 0.05%, 0.025%, 0.01%, and 0.005%. After the HeLa cells were fixed with 4% paraformaldehyde, the concentration of Triton-X 100 decreased to 0.01% and 0.005% for the first time, then, after staining with p53, the mitochondria were stained with 0.1% Triton-X 100 for the second time.ResultsThe expression of p53 was up-regulated (P<0.01) after hypoxia, which could be used in the following immunofluorescence experiment. The co-localization of p53 and mitochondria was observed in the nucleus and cytoplasm in both the methanol group and the mixed solution group. The co-localization of p53 was the most obvious in the mixed solution group. After using 0.1% Triton-X 100, the p53 signal was mainly in the nucleus, but no co-localization was observed. After fixation with 4% paraformaldehyde, to some extent, the reduced concentration of 0.05% and 0.025% Triton-X 100 weakened the p53 signal in nucleus and enhanced the co-localization signal. However, the signal in nucleus was still stronger than that in cytoplasm. When it was reduced to 0.01% and 0.005%, p53 signal was detected in cytoplasm but not in nucleus, suggesting that the nuclear membrane was not penetrated under this condition, but it also failed to penetrate the mitochondrial membrane, leading to the failure of mitochondrial labeling. The second permeability completely avoided the p53 signal in nucleus, and successfully labeled mitochondria, and the co-localization of p53 and mitochondria was detected.ConclusionsCo-localization of p53 and mitochondria is detectable in cells fixed by methanol or methanol and acetone mixture which brings out better results. Penetrating twice with Triton X-100 of different concentrations following paraformaldehyde fixation help avoid signals in nuclei and falicitate co-localization detection.