Vitamin D And Prevention Of Prostate Cancers Biology Essay
Vitamin D is a fat soluble vitamin involved in keeping Ca hemostasis in the organic structure and doing soaking up of Ca from the intestine. It besides has some antiproliferative effects on prostate malignant neoplastic disease cells. In this experiment we have made an attempt to turn out this antiproliferative consequence of vitamin D on prostate malignant neoplastic disease cells. Therefore cell civilization, RNA synthesis and polymerase concatenation reaction ( PCR ) elaboration was done. Knowing that Nuclear vitamin-D receptors ( nVDR ) receptor have important function in cell growing and androgen recptors ( AR ) receptor have proliferative function in prostatic malignant neoplastic disease. From our PCR elaboration, it is seen that AR cistron is more uttered as comparison to nVDR cistron in both treated and control sample. Therefore turn outing that vitamin D has up regulated AR more than nVDR.
It is hydroxylated in the liver into 25 ( OH ) vitamin D and in kidney into 1, 25 ( OH ) 2 vitamin D ( Weisman 2010 ) . This is active signifier or metabolite of vitamin D that enters the cells and binds to its several receptor, that is vitamin D receptor ( VDR ) and so later to its cistron which is a Ca binding protein ( Weisman 2010 ) . After treating the protein such as osteocalcin or Ca binding protein is formed ( Weisman 2010 ) . This protein is responsible for doing Ca soaking up from intestine ( Weisman 2010 ) . Vitamin D is of import for musculoskeletal system. It besides has function in excess skeletal tissues ( Wang et al. 2010 ) . Parathyroid endocrine stimulate the production of 1, 25 ( OH ) 2 VD and Ca decreases its production ( Weisman 2010 ) .
The lack of vitamin D is seen in premature babies, aged people, corpulent people and those enduring from malabsorption ( Weisman 2010 ) . RICKETS and OSTEOMALACIA caused by vitamin D lack in kids and grownups severally ( Weisman 2010 ) . Peoples with lack of vitamin D are more prone to viral respiratory infections ( CANNELL et al. 2006 ) .
Vitamin D lack lead multiple upsets like increased the blood force per unit area, weariness, malignant neoplastic disease, delicate castanetss ( R. Zhang et Al. 2010 ) . Taking vitamin D addendum by those who are enduring from its lack lead to decreased parathyroid endocrine degree, therefore increasing bone denseness and going less prone to cram break ( Van Den Bergh et Al. 2011 ) . The active metabolite that is 1, 25 ( OH ) 2 vitamin D has antiproliferative effects and therefore it down regulates inflammatory marker ( Weisman 2010 ) .
Surveies about function of vitamin D3 in prostate malignant neoplastic disease have revealed that vitamin D3 inhibits growing of malignant neoplastic disease cell in prostate secretory organ and this function is androgen dependant. ( Murthy et Al. 2005 ) .
Prostate is a secretory organ of male generative system and prostate malignant neoplastic disease is a signifier of malignant neoplastic disease that occurs in prostate secretory organ. Age, familial factor, diet and hormonal factor contribute to hazards for developing prostatic malignant neoplastic disease ( Ramis et al. 2011 ) . Prostate malignant neoplastic disease can be diagnose by physical scrutiny like digital scrutiny, raised degree of prostate specific antigen ( PSA ) in the blood and finally by making a biopsy and analyzing it under the microscope ( Kristal et al. 2010 ) . Surveies of vitamin D on prostate malignant neoplastic disease have shown its anti proliferative, antimetastatic consequence ( Schwartz 2009 ) . Furthermore normal prostate cell are besides capable of synthesising 1,25 ( OH ) 2 D. While handling prostate malignant neoplastic disease with vitamin D we should hold cheque of Serum Ca degree, sing hypercelcemia to be a major hazard factor ( Schwartz 2009 ) .
Method and Material
On twenty-four hours one trypzinisation was done by taking spent cell civilization medium and washed with monolayer cells utilizing phosphate buffer solution ( PBS ) . After adding attempt psin to PBS solution, flask was rotated and incubated at 37 grade centigrade for 5 proceedingss and so turning cells were observed under microscope. Then cell civilization medium was added easy to demobilize tryp wickedness. The whole solution put into falcon tubing and centrifuged for 5 proceedingss. The supernatant was removed and the pellet was resuspended with fresh cell civilization. Then 40 micro litter from this solution was taken into eppendrof tubing and the staying solution was placed on ice for seeding cell after cell numeration.
Into eppendorf 40 micro litter suspension and 40 micro litter of trypan blue ( to color the dead cell ) was added and mixed by soft pipetting. Heamocytometer was prepared by washing screen faux pas with our breath. Two bead of cell suspension were added by capillary force on each side and seen under microscope.
3’A’square were counted
Entire cells in3A sq=176
= 58.6 cell/Asq
Number of cell * dilution factor /0.001
=11.7*105 *4ml ( initial media concentration )
Cell concentration=1.17*106 cell/ml
=42 micro litter
Therefore 2958 micro litter of medium + 42micro litter cells.
Since we have to fix 7wells alternatively of 6 Wellss to maintain some extra so, 2958*7= 20.7 milliliters media
=294 micro litter of cell
Therefore we take 3ml/well.
Vitamin D intervention
A 6well civilization home base was prepared by reseeding 50000 cell to each well. Therefore 3ml of concluding volume as calculated above was added to each well. Then vitamin D3 solubilised with 99 % Ethanol hydrated oxide ( EtOH ) to a concentration of 10-7 was added to 3 Wellss. A prepared stock solution of 10-3 nanometer was added to three control Wellss. Besides EtOH of same volume was added to three controls good because vitamin D added to prior 3 good was treated with EtOH. So we have to see that any alteration in the figure of cell is ether due to vitamin D or due to EtOH.
Then civilization home base was incubated for 42hr.
Again trypzinisation was done like twenty-four hours 1 but now 1ml of trypsin and civilization media each alternatively of 3ml were taken and incubated at 37degree centigrade for 5 proceedingss. From three treated home bases solution was put in 1 tubing and labelled as treated. Similarly solution from three control well was taken in another tubing and labelled and centrifuged. The supernatant was removed and pellet was resuspended. Then 40 micro litter of this solution was assorted with 40 micro litter of trypan blue in eppendorf tubing. Haemocytometer was prepared. Now cell were counted in 9Asq.
Treated cells in 9Asq=4/9
Number of cell * dilation factor/0.0001
=8.8*103 * 2ml
Cell in control slide in 9Asq = 9/9
=1 cell/ A sq
1*2ml/0.0001= 20000 cells/ml
20,000 * initial media concentration
= 4*104 cells
( Here we have counted cells in more squares than day1 because now cell are treated and there count is reduced so we need to see cells in more squares. )
From above computation we see treated cell count is reduced as comparison to cell in control solution.
RNAase free environment was maintained. RNA readying was done by utilizing lab manual and QIAGEN kit. RNA was prepared by making cell lysis, cell homogenation in QIA shredder and than edge with RNA filter and so undergoing several rinsing stairss and finally elution stairss was done and RNA was prepaid.
Finding of RNA QUANTIFICATION
Vitamin D 1.7ng/micro litter
Control 4.7ng/micro litter
Average RNA quantification of all groups for control is 6.5ng and for treated is 3.4ng. Treated RNA is less than control because we have less figure of cell in treated Wellss. Therefore RNA quantification consequence supports our cell count consequence. We have hypothesized that vitamin D decreases cell growing and induced programmed cell death. Therefore our hypothesis is supported by cell count and RNA quantification.
Rearward RNA polymerase -PCR synthesize complementary DNA. RNAase free environment was maintained 2RT-PCR reaction was run, for control and treated severally. Material for doing chief mix to fix complementary DNA is as follows.
2*RT Buffer=10 micro litter
2*RT enzyme=1 micro litter
Nuclease free H2O =6 micro litter
Control= 3 Micro litter
Entire = 20 micro litter
2*RT Buffer=10 micro litter
2*RT enzyme=1 micro litter
Nuclease free H2O =4 micro litter
Vitamin D treated= 5 Micro litter
Entire = 20 micro litter
( We have borrowed RNA from other group holding concentration in control 7.1 and in treated 10.4 because we have to fix 50ng /cDNA reaction and our RNA measure is less. If we use our ain RNA so we require more than 9micro litter of RNA and we have to fix a sum of 20 micro litter master mix. Therefore we have to utilize RNA from the above mentioned group )
RT-PCR maestro mix is prepared assorted exhaustively by easy pipetting up and down. Then PCR tubings are placed in pre heated thermic cycler at 37 & A ; deg ; C for 60 proceedingss and so heated to demobilize rearward RNA polymerase for 5 proceedingss at 95 & A ; deg ; C because we do n’t desire the enzyme to be activated once more and so kept the tubings on eyes at 4 & A ; deg ; C.
Step2 PCR reaction
We require chief mix for 4 reactions but prepare chief mix for 5 reaction to avoid pipetting fluctuations.
dNTP0 =5micro litter
Deoxyribonucleic acid polymerase=1 micro litter
10*PCR buffer=12.5 micro litter
Nuclease free water=85 micro litter
Total= 85 micro litter
GAPDH-F=1 micro litter size of GAPDH is 600 base brace
AR-F=1micro litter size of AR is 200 base brace
VDR-F=2micro litter size of VDR is 350 base brace
CDNA-control= 2 micro litter
CDNA-treated=2 micro litter
PCR was run harmonizing to status given in manual.
Agarose gel was prepared by utilizing stuff as fallow
1*TAE Buffer – cataphoresis
Ethimide bromide- discolorations DNA
We have succeeded with treated but non with control PCR good elaboration of GAPDH and AR is seen but VDR shows swoon sets. VDR sets are weaker than GAPDH and AR sets which mean that VDR is less expressed.
Decision and Discussion
All managed to acquire PCR merchandise with good consequence and right size. All managed to do complementary DNA. All manage to pull out RNA. We did non see any alteration in cistron look because it might be that we have incubated cells for less clip. Sing VDR, shows weaker sets because we have stopped PCR at 28 rhythms if these rhythms were allowed to go on than VDR would hold expressed as strong set.