Search or add a thesis

Advanced Search (Beta)
Home > Establishment of Stable Cell Lines Expressing Proteins of Hcv Genotype 3A Pakistani Isolate and Their Molecular Characterization

Establishment of Stable Cell Lines Expressing Proteins of Hcv Genotype 3A Pakistani Isolate and Their Molecular Characterization

Thesis Info

Access Option

External Link

Author

Butt, Sadia

Program

PhD

Institute

University of the Punjab

City

Lahore

Province

Punjab

Country

Pakistan

Thesis Completing Year

2013

Thesis Completion Status

Completed

Subject

Molecular Biology

Language

English

Link

http://prr.hec.gov.pk/jspui/bitstream/123456789/9765/1/Sadia_Butt_Molecular_Biology_Univ_of_the%20Punjab_2013_HEC_Scholar_Received%20date%2028.01.2016.pdf

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676726090219

Asian Research Index Whatsapp Chanel
Asian Research Index Whatsapp Chanel

Join our Whatsapp Channel to get regular updates.

Similar


Hepatitis C virus (HCV) infection is the leading cause of chronic hepatitis which progresses to hepatocellular carcinoma (HCC) afflicting > 170 million people worldwide. HCV 3a is the most common genotype (about 70% of all genotypes) circulating in Pakistan. Expression of HCV individual gene of 3a would facilitate therapeutic and vaccines strategies against chronic HCV and liver Cirrhosis. The aim of the present study was the establishment of stable Huh-7 cell lines expressing structural and non structural proteins of HCV Genotype 3a Pakistani isolate obtained from chronic HCV patients. Blood samples were obtained from chronic HCV-3a positive patients. HCV individual genes were amplified using PCR with gene specific primers having restriction sites and kozak sequence. These gene amplicons were cloned in TA and mammalian expression vector PcDNA3.1+. Restriction digestion analysis and sequencing results confirmed the successful cloning of HCV genes in PcDNA3.1 vector. Huh-7 cell lines were transfected with these constructed plasmids having structural or non-structural HCV genes in confluent cells with lipofectamine. Positive clones were selected with G418 and then confirmed by genome PCR. Subsequently, transcription and expression of the integrated genes were demonstrated by RT-PCR, sequencing and Western blot analysis. We successfully cloned and expressed seven HCV-3a genes in PcDNA3.1 mammalian expression vector. Results of western blot and sequencing PCR confirmed the stable expression of these seven genes. Various cytokines like Tumor necrosis factor alpha (TNF-α), transforming growth factor beta (TGF- β), leptin and Angiotensin were used as indicator of disease progression in huh-7 cells stably expressing HCV structural and nonstructural proteins in vitro. Collectively, these observations suggest a significant involvement of HCV huh-7/Core, huh-7/E2 and huh-7/NS5A in the up-regulation of gene expression proinflammatory cytokine TNF-α and profibrotic TGF-β. No significant change in the expression of Angiotensin-II in vitro was observed. Up-regulation of leptin was observed in huh-7/E2 cell line only. The stable cell-lines expressing HCV-3a individual genes would be a useful tool to investigate the role of various HCV proteins on HCV disease outcome and testing of new therapeutic strategies against HCV.
Loading...
Loading...

Similar Books

Loading...

Similar Chapters

Loading...

Similar News

Loading...

Similar Articles

Loading...

Similar Article Headings

Loading...

متن اور تدوین متن

متنی تنقید :
انسائیکلو پیڈیا ’ امریکانا ‘ نے متنی تنقید کی تعریف کرتے ہوئے لکھا ہے :
’’ متن کے اصل الفاظ کے تعین، اسے مکمل کرنے اور واقفیت واصلیت تلاش کرنے کی غرض سے پرانی تحریروں کے سائینٹفک مطالعے کو متنی تنقید کہتے ہیں۔ ‘‘
متنی تنقید کا اصل مقصد حتیٰ الامکان متن کو اصل روپ میں دوبارہ حاصل کرنا ہوتا ہے۔ اصل روپ سے مراد وہ شکل وصورت ہے جو متن کا مصنف اپنی تحریر کو دینا چاہتا تھا۔ یعنی اگر متنی نقاد کو مصنف کے ہاتھ کا لکھا ہوا نسخہ ملا ہے تو اسے متنی نقاد من وعن ہی شائع نہیں کرسکتا کیونکہ ممکن ہے مصنف سے کچھ الفاظ چھوٹ گئے ہوں یا کچھ الفاظ دوبارہ لکھ دئیے گئے ہوں یا اس قسم کی کوئی اور غلطی ہوئی ہو۔ ایسی صورت میں متنی نقاد کا فرض ہے کہ متن کو ان غلطیوں سے پاک کرے۔ متن کے لیے ضروری ہے کہ بامعنی ہو، اگر سینکڑوں برس کے عرصے میں نقل در نقل کی وجہ سے متن مسخ ہوگیا ہے تو اس کے اصل معنی کا تعین کیا جاسکے۔
متنی تنقید/تنقیدِ متن کے مدارج :
۱۔ تیاری ۲۔ مواد کی فراہمی
۳۔ متن کی تصحیح ۴۔ قیاسی تصحیح
۵۔ اعلیٰ تنقید
۱۔ تیاری :
الف۔مختلف عہد کے نسخے پڑھنا :
متنی نقاد کا فرض ہے کہ مختلف عہد کی تحریروں پر عبور حاصل کرنے کے لیے ان عہدوں کے نسخے پڑھے تاکہ تحریر کی شناخت کے ساتھ ساتھ اس عہد کے الفاظ وتحریر پر اسے عبور حاصل ہوسکے۔ متنی نقاد کو اس عہد سے قبل کے کچھ نسخے بھی پڑھنے چاہئیں۔ اس انتخاب کے باقاعدہ اصول تو نہیں ہیں لیکن اس عہد میں جولوگ ادب پر چھائے ہوں ان میں سے نمایاں لوگوں کو منتخب کرلیا جائے۔
ب۔مختلف عہد کی زبان پر عبور...

تفسیر قرآن میں ام المؤمنین سیدۃ عائشه کا مقام

Quran is the absolute and error free source of knowledge for all mankind. The words and meanings of the Quran both have been revealed by Allah and will remain unchanged for ever. The holy Quran was explained by the Holy prophet and by sahaba as well. Later on different scholars of Islam have made notable contribution in this regard. Many companions of the Holy prophet are famous in the explanation of the Holy Quran. Although Syyeda Aeshah is famous in the field of Hadith but she is one of most prominent Mufassrah of the Quran too. She has deep and correct knowledge of the Holy Quran. In this Article the status of Sayyedah Aesha in the field of tafseer has been discussed. Hopefully the readers will get useful information from this Article

The Effect of Proparanolol on Hepatic Blood Flow in Reducing the Toxicity of Hepatotoxic Drugs Used in the Treatmetn of Chronic Diseases

Tuberculosis, diabetes and epilepsy are the diseases requiring prolonged treatment by certain drugs. Unfortunately their toxic effects sometimes decline the standards of life of the patients in terms of physical and psychological well being. Hepatic toxicity of rifampin, phenytoin, sulfonylureas and carbamazepine cannot be neglected in this regard. These drugs may reduce the efficiency of liver to a large extent. Therefore it is important to consider an adjuvant for this purpose as these drugs cannot be compromised in term of doses, dosage forms or responses. The study has been planned to see the beneficial role of altering liver blood flow in reducing the toxicity of drugs. Propranolol is a non selective beta adrenergic receptor antagonist which reduces the hepatic blood flow. It is scientific to believe that it can reduce the amount of hepatotoxic drug in liver by reducing the hepatic blood flow thereby reducing the DILI. The hepatotoxicity of these drugs with or without propranolol in term of anatomical changes, enzymatic assay, qualitative and quantitative histopathological studies and electron microscopy were evaluated. In this study significant number of healthy rabbits were used which will be divided into nine different groups from A to H. Calculated dose of all four drugs were given to the assigned groups of rabbits with or without propranolol as per dosing schedule. Group A was considered as control and received distilled water only while group B and C were received CBZ alone and in combination of propranolol, respectively. Liver function test and histological evaluation by H and E staining and scanning electron microscopy (SEM) were carried at the end of dosing by using standard procedures. Serum level of ALT, ALP, γGT and bilirubin were significantly (p<0.05) increased in CBZ treated group as compared to control while not significantly elevated in CBZ plus propranolol treated groups. But significant reduction in hepatic parameters was seen in CBZ plus propranolol vi group as compared to CBZ alone. The histopathological examination revealed various features of hepatic architecture damage in CBZ treated group. These results were also supported by micrometry and SEM. The hepatic damage induced by CBZ was successfully ameliorated by propranolol. The hepatic architecture was effectively recovered in propranolol and CBZ treated group which showed in Hand E staining and SEM. Group D and E received rifampicin (RIF) alone and in combination of propranolol respectively. Similar parameters were used to explore the hepatoxic effects of RIF and protection was given by propranolol. Results of liver function test revealed that RIF significantly elevate the serum levels of ALT, ALP, γGT and bilirubin as compared to control. These levels were also higher in RIF plus propranolol treated group but when comparing the levels in between group D and E it was illustrated that propranolol provide significant protection to the RIF induced damage. Histology and SEM of liver sections also supported these results. Liver damage induced by RIF expressed as central vein dilation, infiltration of inflammatory cells, portal vein dilation and damage of hepatocytes. Micrometry revealed that number of viable hepatocytes, their diameter and nuclear diameter were altered.SEM micrograph showed distorted and swollen hepatic cords. All of these changes successfully turned to normal by combined administration of propranolol. propranolol successfully improve the hepatic architecture proved by both qualitative and quantitative microscopy. Glibenclamide (GLB) alone or in combination of propraranolol was administered in Group F and G respectively. Serum levels of ALT, ALP, γGT and bilirubin were estimated and compared in both groups. Results showed that GLB caused significant elevation of liver functions as compared to control. There values of ALT and ALP were significantly high in vii group G as compare to control.When comparing these levels between group G and F significant elevations were seen in group F which showed that propranolol reduced the level of serum ALT, ALP, γGT and bilirubin when administered with GLB. The granuloma and necrosis seen in GLB treated liver was not seen in group G rabbit’s liver. The number of viable hepatocytes and their nuclear diameter which were considerably reduced by GLB were effectively preserved to normal after administering propranolol as adjuvant. Thus histological evaluation of liver tissue through H and E staining and SEM showed that combined administration of GLB and propranolol is helpful in reducing the GLB induced liver damage. Group H and I received phenytoin (PHT) alone and in combination with propranolol, respectively. Evaluation of hepatic functions test expressed that the serum levels of ALT, ALP, γGT and bilirubin were significantly raised in PHT treated group. Serum levels of ALT, ALP, γGT and bilirubin were also significantly high in PHT and propranolol treated group as compared to control. When comparing the values between these two groups it was illustrated that propranolol offered significant protection to the liver. Histological examination of Hand E stained liver tissue showed that PHT caused severe hepatic damage expressed as necrosis, hemorrhage and dilation of sinusoids, inflammation and dilation and congestion of portal vein. Combined administration of propranolol and PHT reduced these changes induced by PHT alone. The micrometric estimation of H and E stained section of live supported the above mentioned results.Minor inflammatory cells were seen and necrosis is absent in liver section of group I rabbits. SEM of liver of group H also showed damage and ruptured hepatic cords with cellular swelling. Protection provided by viii propranolol also expressed through this technique. Propranolol improved the hepatic architecture clearly seen in the SEM micrograph of group I rabbit. It is summarized that all of the aforementioned drugs produced threatened effect on the liver. Propranolol is an approved treatment of portal hypertension and esophageal varices in cirrhotic patient. This effect of propranolol is due to reduction in hepatic blood flow. Propranolol offered beneficial effect in drug induced hepatoxicity may be due to its diminution of portal blood flow thus reducing the supply of noxious substance to the liver. Propranolol may also ameliorate drug induced liver disease due to its affect on cytochrome P450 or due to owing antioxidant action.