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Study on the Imitating Androgen Therapeutic Effects of Buddleja Officinali on Induced Dry Eye in Castrated Rats

Author: WangFang
Tutor: PengQingHua
School: Hunan University of Traditional Chinese Medicine
Course: Integrative Medicine Ophthalmology
Keywords: Buddleia extract Alcohol precipitation water Flavonoids 80% Castrate Dry eye SIT BUT Corneal staining AR bcl-2 mRNA Bax mRNA Lacrimal gland epithelial cells Culture STAT1 phosphorylation Flutamide
CLC: R285.5
Type: PhD thesis
Year: 2009
Downloads: 120
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Abstract


The first part Buddleia Total Flavonoids preliminary optimization studies and preparation of the study medication Objective: To find the best Buddleia flavonoids extraction method, so that the total flavonoid content higher than 80%. Methods: Ethanol water precipitation method, after the use of macroporous resin gradient elution method, in a number of experiments to explore alcohol water sink water multiples, and macroporous resin elution conditions, to determine the most flavonoids Buddleia Jia extraction process. Results: The two small test that determines extraction process. Take Buddleia herbs 15kg extraction, to be finished 110g. Two parallel samples removed from the finished product, for HPLC, and the results have been seen Linarin basic impurities are eluted to Linarin already prepared standard solution as a control, using the external standard method, linarin content 17.573% and 17.011%, 17.292% average. Have been identified through Linarin Purple: outside the standard curve, a total flavonoid content of 81.488%. Conclusion: After ethanol precipitation water by macroporous resin gradient elution method is the extraction of total flavonoids Buddleia preferred method, this method can extract the material content is higher than 80%, in line with standard V class of drugs, can be used for the study. The second part Buddleia flavonoids intervention castrated male lacrimal gland causes dry eye BaxmRNA, bcl-2 mRNA and androgen receptor expression Objective: To establish rat androgen levels decrease dry eye model; observe the lacrimal gland AR organization raised the case of an initial evaluation of Buddleia androgenic effects of flavonoids intended; observe basic tear secretion and tear film stability, TEM lacrimal gland ultrastructural changes; observation Buddleia flavonoids on androgen levels drop Apoptosis induced dry eye have inhibitory effect. Methods: Healthy one-month-old male Wistar rats were 150, weighing about 0.2kg. Were randomly divided into A1, B1, C1, D1, E1, A3, B3, C3, D3, E3, A5, B5, C5, D5, E5 total of 15 groups of 10. A representative of the normal group; B represents the sham group; C represents the model group; D represents androgen control treatment group; E represents Buddleia flavonoid treatment group; 1 represents feeding a month; 3 represents rearing three months; 5 represents Feeding five months. Castration method used to establish the decrease in androgen levels animal models of dry eye. Animals in each group by the same person checks, each animal each inspection time, location, lighting levels, humidity, and at the same temperature. Before modeling, respectively, after the completion of the last administration, Schirmer I test (SIT) and the tear film breakup time (BUT) examination. In SIT, BUT the difference with the normal group formation, corneal staining denoted as () above as abnormal. Results: 1. Corneal staining: A, B group, corneal fluorescein staining did not change significantly; C group, four months later, the corneal epithelial defect started, fluorescein staining was positive and increased with time; D, E groups were 4,6 months after gavage, corneal fluorescein staining point reduction. Two in each group SIT measurements: C group (except group C1 outside) filter paper wet length shortened, and castration before the difference was significant (P lt; 0.05), with time the difference was not statistically significant (P gt; 0.05). Group D (D1 group except outside) long wet filter paper increased significantly compared with C group, the difference was statistically significant (P lt; 0.05). Group E (E1 group except outside) long wet filter paper increased significantly compared with C group, the difference was statistically significant (P lt; 0.05). Group D5 and E5 group difference was statistically significant (P lt; 0.05). Three in each group BUT measurements: C (except C1 outside the group) group tear film breakup time is shortened, and castration before the difference was significant (P lt; 0.05), and with time the difference was statistically significant (P lt; 0.05 ). Group D (D1 group except outside) tear film break-up time increased significantly compared with C group, the difference was statistically significant (P lt; 0.05). Group E (E1 group except outside) tear film break-up time increased significantly compared with C group, the difference was statistically significant (P lt; 0.05). Group D5 and E5 group difference was statistically significant (P lt; 0.05). These two indicators of changes in animal models of dry eye explanation success; Buddleia flavonoids on androgen levels decrease dry eye have a therapeutic effect, but with the duration of the extension reduces healing, and androgen treatment differences . 4 SEM results showed that: Buddleia flavonoids treatment group compared with the model group organizational structure lacrimal neat, necrosis, degeneration of tissue reduction, reduced apoptosis, inflammatory cells gradually subside. However, with the duration of the extension and its role diminished, and with the formation of androgen effects difference. 5.AR and bcl-2 mRNA expression showed: C group (except group C1 outside) AR and bcl-2 mRNA expression was increased, and the difference before castration significantly (P lt; 0.01), and with time the difference was statistically significant (P lt; 0.01). Group D (D1 group except outside) increased significantly compared with the C group, the difference was statistically significant (P lt; 0.01). E group (except group C1 outside) increased significantly compared with the C group, the difference was statistically significant (P lt; 0.05). Group D5 and E5 group difference was statistically significant (P lt; 0.01). 6. Bax mRNA expression showed: C group (except group C1 outside) Bax mRNA expression was increased, and the difference before castration significantly (P lt; 0.01), and with time the difference was significant (P lt; 0.01). Group D (D1 group except outside) increased significantly compared with the C group, the difference was statistically significant (P lt; 0.01). E group (except group C1 outside) increased significantly compared with the C group, the difference was statistically significant (P lt; 0.05). Group D5 and E5 group difference was statistically significant (P lt; 0.01). These results suggest that flavonoids Buddleia can upregulate the lacrimal gland in AR, the increase in the number of AR, but with the duration of the extension, which raised weakened, and with the upregulation of androgen formation differences; make the bcl-2 mRNA in the lacrimal gland increased expression of Bax mRNA expression decreased, but with the duration of the extension and its role weaker androgens. Conclusions: 1. Using an improved method of castration successfully established decrease androgen levels in animal models of dry eye. This method is less damage in rats, simple, and laid the foundation for further study. Two rat lacrimal gland tissue in androgen receptors. 3 Buddleia flavonoids increased androgen levels may decrease dry eye lacrimal gland tissue expression of androgen receptor, testosterone propionate produce the same effect, but increases with the duration of the extension of its weakened, and male effect of hormones significant difference formation. 4 Buddleia flavonoids decrease androgen levels in the treatment of dry eye may be related to a mechanism to be generated after androgen effect on apoptosis-related gene bcl-2 mRNA upregulation and downregulation of expression of Bax mRNA. The third part Buddleia flavonoids drug-containing plasma cells interfere with dry eye model AR mRNA expression and protein expression of STAT1 phosphorylation study of the impact Objective: To establish the decrease in androgen levels in a cell model of dry eye; fumble drug-containing plasma optimum amount of interference; observation of drug-containing plasma lacrimal gland epithelial cells on expression of AR; investigate if the drug containing plasma and AR binding, signal transduction pathway for the activation of STAT1; application of androgen receptor blockers in the same Methods to detect the expression of AR mRNA expression, and protein expression of STAT1 phosphorylation to determine Buddleia flavonoids drug containing plasma is combined with the AR, but not other receptor binding effect occurs. Methods: 120 a month, weight 150g-180g healthy male Wistar rats orbital lacrimal gland, lacrimal gland were isolated and cultured epithelial cells. To H2O2-induced rat lacrimal gland epithelial cell apoptosis, decrease androgen levels established apoptosis dry eye condition. Preparation of drug-containing plasma cells by MTT method fumble intervention best content; while the establishment of blank plasma group Buddleia flavonoids drug-containing plasma intervention group, testosterone propionate intervention group were observed in each group AR expression and phosphorylation of STAT1 situation; and applying the androgen receptor blocker flutamide, study Buddleia flavonoids intended androgenic effects. Results: 1.10d to see the culture of lacrimal gland epithelial cells were round or oval cell body hypertrophy and bright, located in the central nucleus, membrane clear, grow more active. 2.MTT assay showed that the best intervention drug-containing plasma volume 8.95 × 10-2. 3. Western blot results showed that after the intervention of drug-containing plasma, Buddleia flavonoids drug-containing plasma in the intervention group increased expression of AR, and The difference between the control group was significant (P lt; 0.01); testosterone propionate intervention group AR protein expression increased and the difference between the control group was significant, with Buddleia flavonoids intervention group compared to the drug-containing plasma the difference was statistically significant (P lt; 0.01). Buddleia flavonoids drug-containing plasma intervention group increased the expression of STAT1, the intervention group compared with the blank plasma difference was significant (P lt; 0.01); Buddleia flavonoids drug-containing plasma in the intervention group compared with STAT1 expression Testosterone propionate low expression level of the intervention group, but the difference between them was significant (P lt; 0.01). Intervention in each group of drug-containing plasma androgen receptor blocker added after each group of AR expression and phosphorylation of STAT1 no difference between. Conclusions: 1. Using improved collagenase Ⅱ and repeated adherent successfully isolated and cultured in vitro rat lacrimal gland epithelial cells. This method improves the purity of the lacrimal gland epithelial cells, methods and practical, laid the foundation for further study. 2 lacrimal gland epithelial cells in the presence of androgen receptors. Buddleia flavonoids drug-containing plasma lacrimal gland epithelial cells with a combination of AR and not with other receptors on lacrimal gland epithelial cells produce AR upregulation, androgenic effects occurred proposed. 3 Buddleia flavonoids drug-containing plasma can be combined with the promotion of AR expression of STAT1 phosphorylation and activation of STAT1 cell signal transduction pathways, which produce testosterone propionate same androgenic effects.

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CLC: > Medicine, health > Chinese Medicine > Of Pharmacy > Pharmacology > Chinese medicine Experimental Pharmacology
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