人類脂肪幹細胞培養於電漿處理幾丁聚醣薄膜之初步研究
人類的脂肪幹細胞,取得容易,且來源不涉及倫理問題,是一種理想的城體細胞來源,目前已發現其可分化成骨細胞,軟骨細胞,脂肪細胞,等等,因此深具未來發展的潛力,將可應用於組織工程細胞治療上,而Chitosan(幾丁聚醣)則是一種具生物相容性,生物分解性且無生物毒性的材料,在生醫材料的領域一具相當發展性,在這個實驗中,我們將chitosan薄膜經過氬氣電漿處哩,並測量其與水的接觸角.發現電漿處理時間越長,接觸角越小,表示電漿處理有助於增加chotosan薄膜表面的親水性.細胞在經過電漿處理的薄膜上,其黏附情形一隨處理時間增長,黏著數目越多,且形態愈扁平,表示電漿處理過的chotosan表面有助於細胞的吸附.;Human Processed Lipoaspirate cells (hPLA) can be subtracted easily from lipoaspirate and the source of the cells does not violate the moral and ethic standard. Therefore, it is an ideal source of somatic stem cells. Recently, researches show that the hPLA cells have the ability to differentiate into osteoblast cells, chondrocytes cells, adipose cells, and skeletal muscle cells. In the future, they have great potential in tissue engineering or cell therapy. Chitosan is a biocompatible, biodegradable, and non-toxic material. It is also an advanced material to be used in the biomaterial field. In this study, we treated chitosan film with argon plasma and measured its contact angle with water. The contact angles decreased as the duration of plasma treatment on chitosan increased, indicating that plasma treatment has a positive influence on increasing the hydrophilicity of chitosan film surface. Moreover, the numbers of hPLA cells adhering to chitosan films increased and their morphology became flatter when the durations of plasma treatment on chitosan films prolonged. Hence, the result showed that plasma treatment on chitosan films also promoted the adhesion of hPLA cells on chitosan film surfaces.
Finding a Better Brain Booster
The purpose of my project was to determine which activity improved academic performance the most: 10 minutes of exercise, 10 minutes of Brain Gym (cross-lateral movements), or a combination of 5 minutes exercise and 5 minutes Brain Gym. This project was conducted to find which activity would be a better learning aid in grade 4 students. There were many steps to conducting this study. First, I located teachers and classes, willing to allow the testing and determined days and times to test. Next, I created consent forms to explain the project and had permission forms from the students and their parents signed. Then I prepared 15 math and reading tests at students’ grade level. There were two tests for 15 days- labeled Before Test and After Test. When I administered the tests for the classes doing the activities, I made sure the tests were given by the classroom teacher, which kept the students motivated. Tests were given every day for 5 days. The Before Test was given at 1:00 pm. Then at 1:30 pm students did 10 minutes of the planned activity for that week. Immediately after the activity, students were given the After Test. This was done every day and was repeated for Exercise, Brain Gym, and Combination week. The control class was designed to determine if doing the test for a second time in a day improved the student’s performance. The Before Test was given at 1:00 pm and then at 1:30 pm. When the Before Tests were completed, the students continued with their daily work for 10 minutes. Then at 1:40 pm students were given the After Test. This was repeated for Exercise, Brain Gym, and Combination week. Students were given 10 minutes to complete the scheduled activity that week; either Exercises, Brain Gym or a combination of the two. During the week of Exercise, Jumping jacks, Skipping, Stride jumps, Burpies, Twisties and Jogging on the spot were completed in the 10 minutes provided. During the Brain Gym week, there were 26 Brain Gym movements. The lists of movements were rotated each day; so all movements were performed. The test results of this study were intriguing. (Bar=Standard Deviation) I concluded that 10 minutes of exercise was the better brain booster. Exercise improved academic performance by 9.8%. Brain Gym improved academic performance by 0.2% and the combination activities increased test scores by an average of 3.2%. The control class test scores decreased on average by 1.0% indicating that writing the tests twice in a day, did not improve students’ test scores. Exercising for 10 minutes improved student’s math scores by 11.0% and reading scores by 6.9%. Brain Gym math test scores decreased by 1.4%, and increased in reading by 1.8%. In the combination of the two, the math test scores decreased by 1.0%, reading test scores increased by 7.4%. Overall, the most effective and reliable brain booster was 10 minutes of mild aerobic exercise alone.
Expression of TRPV5 in Astrocytes: Implications for Ischemic Stroke
Elevation of intracellular calcium secondary to increased calcium influx along with increased gliosis are implicated in the pathogenesis of focal ischemic stroke. In astrocytes, which play a major role in maintaining homeostasis in brain ischemia, the identities of the ion channels responsible for increased calcium influx during ischemia is relatively unknown although several Ca2+-permeable transient receptor potential (TRP) channels have been identified to have contributing roles. The transient receptor potential vanilloid 5 (TRPV5) channel is a Ca2+-permeable cationic channel expressed primarily in kidney epithelial cells and at low levels in the brain, but the exact localization and role this channel plays in the brain has not been explored. To investigate the possible role TRPV5 plays in astrocytic calcium influx in ischemia, we examined the functional expression of TRPV5 in astrocytes subjected to hypoxia-ischemia in vitro and in rat models of ischemic stroke in vivo. We hypothesize that TRPV5 contributes to increased calcium influx in ischemia. By treating astrocytes with culture conditions without glucose and with low oxygen levels, we found that TRPV5 is upregulated with increasing durations of simulated hypoxia-ischemia in vitro. Similarly, rat models of ischemic stroke with middle cerebral artery occlusion also show TRPV5 upregulation in reactive astrocytes, suggesting a possible role of TRPV5 in reactive gliosis in vivo. Microfluorimetric intracellular calcium imaging using Fura-2 on primary cultured astrocytes show a voltage-independent increase in astrocytic calcium influx after hypoxia-ischemia in vitro that is selective for extracellular Ca2+ concentration and is reduced by inhibition of TRPV5 with ruthenium red. Electrophysiology measurements using the whole-cell patch clamp technique on primary cultured astrocytes reveal a non-selective cation current similar to that of TRPV5 that is inhibited by Mg2+, another inhibitor of TRPV5. Preliminary results on astrocyte cell viability during hypoxia-ischemia with TRPV5 inhibition by ruthenium red also suggest that inhibition of TRPV5 could enhance astrocyte survival and reactive gliosis in vitro, indicating a beneficial role in blocking non-selective Ca2+ entry via TRPV5 into astrocytes. Since TRPV5 is highly selective for Ca2+ and an important channel for Ca2+ absorption in various epithelial cells, TRPV5 upregulation may contribute significantly to elevated Ca2+ influx in astrocytes in hypoxia-ischemia. Also, Ca2+ influx has been demonstrated to play a crucial role in reactive gliosis, further suggesting that TRPV5 upregulation is involved in reactive gliosis. We propose that TRPV5 is involved in ischemia-induced calcium influx in astrocytes, and might participate in the pathogenesis of focal ischemic stroke.
Investigation of the Role of Mammalian Siderophore 2,5-DHBA in Neurodegeneration
Lipocalin 2 (LCN2), a 25-kDa secreted protein that belongs to the lipocalin family, is known to bind to a class of bacterial Fe-binding molecules known as siderophores. Iron is essential for bacterial growth. To obtain iron from host cells, bacteria produce siderophores, such as Enterochelin (Ent), to bind and transport host iron into the bacterial cell. In response, the host produces LCN2 to bind the iron-laden enterochelin, forming the tricomplex, LCN2: Ent: Fe3+. This inhibits bacterial growth as iron has been sequestered by LCN2. Devireddy et.al. 2005, proposed the binding of the tricomplex, LCN2: Ent: Fe3+ with the LCN2 receptor (LCN2R). This resulted in the internalisation of the complex, releasing the bound iron into the cell. The increase of intracellular iron was reported to cause cell mortality. Recent publications postulated 2,5-dihydroxybenzoic acid (2,5-DHBA) to be an endogenous mammalian siderophore homologue in mouse in vivo and in vitro studies, which could sequester LCN2 and iron. High iron concentrations in the brain have been consistently observed in Alzheimer's disease and Parkinson's disease. Accumulation of intracellular iron is known to be toxic to neurons, resulting in neurodegeneration. Hence, this study aims to determine the role of 2,5-DHBA as the mammalian siderophore in a cell culture model of neurodegeneration. We hypothesise that addition of 2,5-DHBA to cells exposed to LCN2 will result in increased iron uptake into neuronal cells, reducing cell viability. SH-SY5Y (human neuroblastoma) cell line was used in our study. To determine if SH-SY5Y is a suitable cell line, endogenous levels of LCN2 and LCN2R mRNA and protein expression were determined using reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot analysis respectively. Preliminary results showed presence of both the LCN2R mRNA and protein but absence of LCN2 mRNA. This could be due to the low expression of LCN2 when not exposed to stress. Hence, to simulate conditions of neurodegeneration (by inducing high expression of LCN2), SH-SY5Y was treated with Kainic Acid (KA). After KA, LCN2 mRNA and protein expression levels will be detected again. With the successful upregulation of LCN2 gene expression, SH-SY5Y will be treated with 2,5-DHBA with KA treatment to determine cell viability using the MTS cell proliferation assay. A decreased cell viability or increased expression of pro-apoptotic genes would support the function of 2,5-DHBA as a mammalian siderophore in the brain. Furthermore, KA treatment can also be applied to microglial or astrocyte cell lines, which are known to secrete high levels of LCN2 when treated with KA. Co-culturing these cells with SH-SY5Y can allow us to study the downstream effects of secreted LCN2 from glial cells binding to the LCN2R receptors on SH-SY5Y neuronal cells. This study will help to further understanding of the relationship between 2,5-DHBA and cellular iron transport. If 2,5-DHBA is able to bind LCN2 and iron to increase intracellular iron levels in the neuronal cells, the formation of the tricomplex, LCN2: 2,5-DHBA: Fe3+, could be targeted for therapeutic interventions in neurodegenerative diseases by reducing intracellular iron levels to help ameliorate the progression of neurodegenerative diseases.
Discovery, Cloning and Recombinant Expression of a Coral Peptide with anti-Bacteria activity
Inflammatory Bowel Disease (IBD) is a prevalent disease of the West which pathogenesis is driven by a combination interaction between bacteria and inflammatory cells. In this study, two Kazal domain peptide from Palythoa Caribaeorum were identified. They were found to exhibit serine protease inhibitory, anti-bacterial effects and low toxicity, making them ideal candidates for IBD treatment due to their ability to inhibit inflammatory cell migration and bacterial load. We amplified their coding DNA sequences via PCR and ligated the resulting PCR product into pGEX-4T3 vector. The recombinant plasmid was verified by sequencing, and restriction digest before being transformed into competent E.coli cells. Following transformation, we induced target peptides expression by IPTG to confirmed successful transformation and peptide production. Selected transformed bacterial colonies were expanded in LB broth before mixing with glycerol and frozen in -80°C freezer to complete the process of cell bank production.