Difference between revisions of "Team:SZU China/Background"

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            <p class="blog-post-meta">January 1, 2014 by <a href="#">Mark</a></p>
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<p>The majority of people may have learned that smoking is a risk factor for lung cancer, but you may not know that almost 50% of the bladder cancers are also related to smoking. As we all know, bladder is a vital organ in our urinary system. However, bladder cancer is listed as the most commonly encountered urinary carcinoma in China and it ranks No.2 in America. According to management goals and prognosis, the bladder cancers are classified as non-muscle-invasive, muscle-invasive, and metastatic.<sup>[1]</sup> Although methods for early diagnosis of bladder cancer have been improved, the 5-year survival rate of metastatic bladder cancer has not been changed significantly<sup>[2]</sup> According to the National Cancer Institute of the United Nation, almost 74,000 people in America will be diagnosed with bladder cancer this year and this number has been increasing for 30 years, which means more and more people will have to face bladder cancer in the future. Therefore, it’s very important for us to come out with an efficient way to cure bladder cancer.</p>
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            <p>Three recombinant plasmids were generated and transiently transfected into a human bladder cancer cell line T24.
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To test the working efficiency of our orthogonal system, we devided the cells into two groups. One has Ack in the culture medium and another does not.
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Luciferase assays were performed using luminometer. Since Ack is essential for our system, we speculated that Renilla Luciferase(Rluc), the output gene in the circuits can be selectively expressed in Experiment group with Ack. As shown in Fig. 1, the activity of Renilla Luciferase(RLUC) varied widely between the two groups, and the circuit only demonstrated significant activity in the experimental group as expected. Therefore, our orthogonal system is verified to be in good condition and can work efficiently.</p>
 
 
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<p>There are many types of treatment for bladder cancer nowadays. Chemotherapy and radiation therapy, for instance, are two major types of treatments.<sup>[3]</sup> However, chemotherapy can not only kill cancer cells, but also healthy cells that grow and divide very quickly, thus creating a considerable side effects such as mouth sore and hair loss. There is also another type of treatment called targeted therapy, which can specifically target at cancer cells with no affect on healthy cells. So this year, we’re trying to construct a gene circuit based on AND GATE and Unnatural Amino Acid (UAA) Orthogonal System to selectively express therapeutic gene in bladder cancer cells to kill them specifically.</p>
 
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            <h3><stromg>Luciferase assay in two plasmids system</strong></h3>
 
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            <p>hTERT and hUPII promoters are cancer specific promoter and bladder specific promoter, respectively. By combining the two promoters into the design, we supposed that the constructed circuits have the ability to selectively identify bladder cancer cells, in which telomerase and uroplakins are both expressed.<br>
 
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To test this hypothesis, we used the luciferase reporter gene as the output of the circuit and tested the luciferase activity in HFC, which is short for human fiber epithelial cell, Hela, a cervical carcinoma cell line and 5637, a bladder cancer cell line. Also, we reconstructed our system in two plasmids to test if two plasmids system can perform higher efficiency.<br>
 
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The result (Fig.2) shows that the activity of LUC in 5637 with Ack was about two times as high as that in 5637 without Ack and three times as high as that in Hela, while it could not be measured in HFC. We can learn from this result that this system is perfectly safe for normal cells, with no luciferase being detected in HFC. However, compared with three plasmids system, whose RLUC activity of experimental group is about 7 times as high as that of control group, the working efficiency of our orthogonal system in two plasmids is much lower. So we had better construct our system in two plasmids to express higher level of therapeutic gene in bladder cancer cells and reduce its expression in other cell types to increase its specificity.</p>
 
 
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<p>1. Falke J, Witjes JA. Contemporary management of low-risk bladder cancer. Nat Rev Urol. 2011;8:42–9.
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2. Lei AQ, Cheng L, Pan CX. Current treatment of metastatic bladder cancer and future directions. Expert RevAnticancer Ther. 2011;11(12):1851–62.
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3. Marta GN, Hanna SA, Gadia R, Correa SF, Silva JL, Carvalho Hde A. The role of radiotherapy in urinary bladder cancer: current status. Int Braz J Urol. 2012;38(2):144–53.
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            <h3><stromg>Green fluorescent light measurement in two and three plasmids system</strong></h3>
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            <p class="blog-post-meta">December 23, 2013 by <a href="#">Jacob</a></p>
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            <p>To further verifying the working efficiency of the unnatural amino acid orthogonal system, we replaced the amber mutated output gene in former plasmids to amber mutated GFP. The plasmid psiCHECKTM2-CMV-GFP was used as a positive control to monitor transfection and expression efficiency. The two plasmids and three plasmids system being constructed by GFP recombinant plasmids were transiently transfected into 5637 and T24, both of which are bladder cancer cell line.
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Pictures of green fluorescent light produced by target cells were taken by fluorescent microscope. From these pictures (Fig. 3), we can see there is no expression of GFP being detected in no Ack groups while the light intensities are rather high in with Ack groups no matter in T24 or 5637 cell lines. These results indicate that the output gene is expressed only in cells which have Ack in the culture medium. These findings suggest that the constructed circuit based on AND GATE and UAA orthogonal system can be used to specifically identify bladder cancer cells, and may yield a new therapeutic approach for bladder cancer.</p>
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            <p class="blog-post-meta">December 14, 2013 by <a href="#">Chris</a></p>
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Revision as of 13:33, 18 September 2015



Background






The majority of people may have learned that smoking is a risk factor for lung cancer, but you may not know that almost 50% of the bladder cancers are also related to smoking. As we all know, bladder is a vital organ in our urinary system. However, bladder cancer is listed as the most commonly encountered urinary carcinoma in China and it ranks No.2 in America. According to management goals and prognosis, the bladder cancers are classified as non-muscle-invasive, muscle-invasive, and metastatic.[1] Although methods for early diagnosis of bladder cancer have been improved, the 5-year survival rate of metastatic bladder cancer has not been changed significantly[2] According to the National Cancer Institute of the United Nation, almost 74,000 people in America will be diagnosed with bladder cancer this year and this number has been increasing for 30 years, which means more and more people will have to face bladder cancer in the future. Therefore, it’s very important for us to come out with an efficient way to cure bladder cancer.




There are many types of treatment for bladder cancer nowadays. Chemotherapy and radiation therapy, for instance, are two major types of treatments.[3] However, chemotherapy can not only kill cancer cells, but also healthy cells that grow and divide very quickly, thus creating a considerable side effects such as mouth sore and hair loss. There is also another type of treatment called targeted therapy, which can specifically target at cancer cells with no affect on healthy cells. So this year, we’re trying to construct a gene circuit based on AND GATE and Unnatural Amino Acid (UAA) Orthogonal System to selectively express therapeutic gene in bladder cancer cells to kill them specifically.





1. Falke J, Witjes JA. Contemporary management of low-risk bladder cancer. Nat Rev Urol. 2011;8:42–9. 2. Lei AQ, Cheng L, Pan CX. Current treatment of metastatic bladder cancer and future directions. Expert RevAnticancer Ther. 2011;11(12):1851–62. 3. Marta GN, Hanna SA, Gadia R, Correa SF, Silva JL, Carvalho Hde A. The role of radiotherapy in urinary bladder cancer: current status. Int Braz J Urol. 2012;38(2):144–53.