Migration Rate of Colo16 Cells Exposed to UV Light

Migration Rate of Colo16 Cells Exposed to UV LightIntroductionCells clear to ultraviolet (UV) rays of light often supress signalling path meanss associated with inflammation as well as the immune system and as a result of activating the p38 mitogen activate protein kinase (MAPK) pathway. Radiation from UV light is from the electromagnetic spectrum and brook be divide into two primary wavelengths UVA 320 400 nm and UVB 290 320 nm. UVA rays are more rife than UVB, however both UVA rays arrive at the subcutaneous layer of skin and UVB rays r apieceing the dermis are cognize to cause the development of skin goatcers via disconfirming the DNA of skin cubicles (1, 2, 3).The effect of UV light on stalls evoke be pass judgmentd by determining the quantify the jail kiosks take to immig graze back to their normal positions afterwards(prenominal) the creation of an artificial shock. An inexpensive and elementary tick to perform to make such measurements is the ding s tudy. The scratch assay uses a pipette tip to create an artificial respite among the mobile phonephoneular telephones infra aseptic conditions and uses high re outcome imaging devices connected to an change microscope to take pictures of the stalls migrating at specialised epoch intervals. The images cornerstone then be used to calculate the withdrawnnesss travelled under the different UV exposures and so a comment on how the cellphones were affected under the different conditions locoweed be made (1, 2, 3, 4).In this experiment, Human squamous cell carcinoma (SSC) cells that are known to hasten a dysfunctional p53 proteins were taken from the epithelial Colo16 cell cable system and used to measure the migration lengths of the cells under different UV exposures. Then the matrix metalloproteinase (MMP) inhibitor GM6001 was added to the Colo16 cells in order to rec all everywhere if it had an effect on the cell migration distances under the same UV exposures (1, 3 , 5, 6).GM6001 is a MMP inhibitor that inhibits the enzyme collagenase which destroys the peptide bonds of the protein collagen found in the extracellular matrix. Collagen is an central component of the connective tissue and helps the cells adhere to their meet surfaces. GM6001 has been shown to block the phosphorylation of the dermal growth factor receptors (EGFR) and inhibit cell migration responses (3, 5).AimsTo determine the migration rate of Colo16 cells unfastened to UV light at intervals of 12 hours over 48 hours after the creation of an artificial wound ( prisonbreak).To determine the effect of the MMP inhibitor GM6001 on the migration rate of Colo16 cells exposed to UV light at intervals of 24 hours over 72 hours after the creation of an artificial wound (gap).HypothesisIf cells from the Colo16 cell bank line are set with the MMP inhibitor GM6001 then it is expected that the mean gap distance will increase when compared with untreated Colo16 cells under the same expos ure to UV light collectible to the inhibition of collagenases that break downhearted collagen and the blocking of EGFR phosphorylation.Materials and methodsAs per the BIOL2299 2014 Prac 4 lab nones.ResultsRaw class data for the untreated and treated Colo16 cell gap distance (mm) is shown in plank 1 and dining table 2 respectively.From the class data our group was Group A shown in Table 3 and Table 4, and the mean class data along with its cadence deviation set is shown in Table 5 to Table 8.Table 1. elucidate untreated Colo16 Cell Gap Distance (mm).Table 2. sectionalisation Colo16 GM6001 Treated Cell Gap Distance (mm).Table 3. Group A Colo16 Untreated Cell Gap Distance (mm).Table 4. Group A GM6001 Treated Colo16 Cell Gap Distance (mm).Table 5. miserly clear Colo16 Untreated Cell Gap Distance (mm).Table 6. Class sample Deviation (SD) of Colo16 Untreated Cell Gap Distance (mm).Table 7. Mean Class GM6001 Treated Colo16 Cell Gap Distance (mm).Table 8. Class Standard Deviation (SD) of GM6001 Treated Colo16 Cell Gap Distance (mm). jut out 1. Line represent showing the mean gap distance of Untreated Colo16 cells under different UV exposures along with the standard deviation determine after the creation of a synthetic gap using a pipette tip at intervals of 12 hours over 48 hours post-irradiation. anatomy 2. Line graph showing the mean gap distance of Colo16 cells treated with the MMP inhibitor GM6001 under different UV exposures along with the standard deviation values after the creation of a synthetic gap using a pipette tip at intervals of 24 hours over 72 hours post-irradiation. backchatFrom the line graph in Fig. 1, the Colo16 cells under UV radiation showed a s depress decline in gap distance compared with the control. And the Colo16 cells exposed to UVA + UVB rays had the pokey decrease in gap distance over 48 hours after the creation of the artificial wound with a gap distance of followly 0.200 mm suggesting that both UVA and UVB work with syne rgy in concert and the p38 MAPK pathway and and then cause a delay in wound mend.From the line graph in Fig. 2, the Colo16 cells treated with the MMP inhibitor GM6001 exposed to different UV conditions had a detain cell migration response when compared to the control GM6001 Colo16 cells that were not exposed to UV irradiation. This whitethorn be due to the inhibition of collagenases that help breakdown the collagen found in the extracellular matrix (ECM) of the Colo16 cells via the action of GM6001.GM6001 is a MMP inhibitor that inhibits the enzyme collagenase that normally is involved in the breakdown of collagen of the ECM. This inhibition may affect the cell migration of Colo16 cells during wound healing as a result of the collagen not universe broken down by the collagenases in the ECM and therefore resulting in the cells being adhered to their surrounding for a longer time increasing the time required to reason the gap and complete the wound healing process.Discussion que stionsWhat other ways are there to determine whether cells are migrating into the wound or proliferating into it? (Are there morphological characteristics of cell migration or proliferation?)Other ways of determining cell migration or proliferation include1 Immunofluorescence staining cells can be dye using fluorescence markers that use antibodies to bind to specific antigens associated with cell migration or proliferation such as the proliferating cell nuclear antigen (PCNA) (7).2 Laser scanning confocal microscopy cells can be viewed under high resolution and the morphological features of migrating cells can be observed such as the formation of fibrin matrices, an increase of length, width and total spread of fibroblasts (7).3 Boyden chamber which measures cell migration via the determination of a chemical substance gradient based on chemicals release via chemotaxis during cell migration (7).What is a Boyden chamber and why would you use it? Give 3 examples of where it can be used.A Boyden chamber is an in vitro chemotaxis assay that measures cell migration via the analysis of leukocyte chemotaxis, where cells are displace in pores at the top of a chamber and allowed to emigrate by dint of its pores containing chemotactic agents. After incubation the cells are stained and the number of cells that have migrated to the lower portion of the chamber (8).The Boyden chamber can be used to measure1 Cell migration based on chemicals released to the cells surrounding, and determine if a specific chemical causes the cells to migrate towards or onward from the arousal (Chemotaxis) (8).2 Cell migration based on the gradient of specific extracellular matrix proteins, and determine if a particular protein causes the cells to migrate towards or away from the protein. This can be done via the coating of the chamber with the protein of choice (Haptotaxis) (8).3 Cell migration rate of tumour cells through the vascular endothelium toward specific chemokines (8). c onstitute 3 limitations of the scratch method.Some limitations of the scratch method include1 Creation of an artificial wound of the same approximate width can be extremely difficult and any variance in the gap distance between the different cells at time 0 hr can greatly affect the significance of the results (4).2 The incubation of the cells after the creation of the wound (scratch) has to be long enough to help the cells migrate during the fastest time frame and any variance in this may lead to the cells not migrating equally, therefore affecting the results. Also, the temperature and environmental conditions of the incubator have to be at their ideal values for healthy cellular migration (4).3 Highly time consuming, in order for the readings to be statistically monumental at least 100 readings of distance for each sample and each experiment must be repeated at least tether times. As a result, this creates copious amounts of data that consumes a lot of time and energy to ana lyse (4).If you are investigating cell growth factors on cell migration, should serum be present in the media? Explain your answer.Yes, serum containing the growth factors should be found in the media in order to lease its effects and the growth factors should be soluble in the serum and added to the media to begin with the addition of cells in order to be equally distributed in the solution (4).What are the main differences in investigating a scratch assay using non-transformed vs transformed cells?Transformed cells are transfected with a plasmid cryptograph the gene of interest along with a plasmid marker in advance the conduction of a scratch assay, whereas non-transformed cells are investigated without being transfected with a specific plasmid (4).Apart from taking photos of the cells over time, propose another way you may be able to show cell migration.Cell migration can be shown via the conduction of an electric cell-substrate impedance sensing (ECIS) in vitro cell migrati on assay that measures cell migration via the creation of a wound by exhalation an electrical current through the cell and causing electroporation. Then cell migration is measured via the calculation of the cells impedance in ohms over a specific time as seen in Fig. 3 (9). throw 3. Typical ECIS data involving the electric cell-substrate impedance sensing (ECIS) in vitro cell migration assay (http//www.biophysics.com/woundhealingpubs.phpECIStheory).Which of the methods is the one that most labs would not use, explain why you animadvert this would be so.Most labs would not use the ECIS in vitro migration assay due to its expensive cost and its relatively time consuming cell type dependant incubation times. Another in vitro migration assay that would not be used in labs is the microfluidics-based system due to its need for nanofabrication facilities and its evenhandedly very expensive cost (4).ConclusionIn conclusion, Colo16 cells under UV radiation showed a slower decline in gap d istance compared with the control. And the Colo16 cells exposed to UVA + UVB rays had the slowest decrease in gap distance over 48 hours after the creation of the artificial wound with a gap distance of only 0.200 mm suggesting that both UVA and UVB work with synergy together and the p38 MAPK pathway and therefore cause a delay in wound healing.Whereas, the Colo16 cells treated with the MMP inhibitor GM6001 exposed to different UV conditions had a delayed cell migration response when compared to the control GM6001 Colo16 cells that were not exposed to UV irradiation. This may be due to the inhibition of collagenases that help breakdown the collagen found in the extracellular matrix (ECM) of the Colo16 cells via the action of GM6001.This inhibition caused by GM6001 may affect the cell migration of Colo16 cells during wound healing as a result of the collagen not being broken down by the collagenases in the ECM and therefore resulting in the cells being adhered to their surrounding fo r a longer time increasing the time required to close the gap and complete the wound healing process.ReferencesBIOL2299 biology of Tissue Growth and Repair Manual, 2014.Reichrath J, Rass K. Ultraviolet damage, DNA repair and vitamin D in nonmelanoma skin cancer and in malignant melanoma an update. Advances in experimental medicine and biology. 2014810208-33.Muthusamy V, Piva TJ. A comparative study of UV-induced cell signalling pathways in human keratinocyte-derived cell lines. Archives of dermatological research. 2013305(9)817-33.Liang CC, Park AY, Guan JL. In vitro scratch assay a convenient and inexpensive method for analysis of cell migration in vitro. Nature protocols. 20072(2)329-33.Grobelny D, Poncz L, Galardy RE. Inhibition of human skin fibroblast collagenase, thermolysin, and Pseudomonas aeruginosa elastase by peptide hydroxamic acids. Biochemistry. 199231(31)7152-4.Moore GE, Merrick SB, Woods LK, Arabasz NM. A Human Squamous Cell Carcinoma Cell Line. Cancer Research. 197 535(10)2684-8.subgenus Chen HC. Boyden chamber assay. Methods in molecular biology (Clifton, NJ). 200529415-22.http//www.cellbiolabs.com/boyden-chamber-assayshttp//www.biophysics.com/woundhealingpubs.phpECIStheory