24 research outputs found
Temperature and physical property control for polymerization reactors
ํ์๋
ผ๋ฌธ(๋ฐ์ฌ)--์์ธ๋ํ๊ต ๋ํ์ :ํํ๊ณตํ๊ณผ,1996.Docto
Construction of efficient expression vector system using cis-acting elements for Lactococcus lactis
ํ์๋
ผ๋ฌธ (์์ฌ)-- ์์ธ๋ํ๊ต ๋ํ์ : ๋์๋ช
๊ณตํ๋ถ, 2014. 2. ์ต์ค์ฌ.Many approaches have been attempted to improve the heterologous protein in various bacteria. Specifically, the plasmid-based expression system has been used to achieve the recombinant protein production as an easy and useful tool to manipulate. There are three strategies to improve the expression level of recombinant protein. Such as 1) introduction of high copies plasmid-based expression vector system, 2) construction of gene multimerization cassette as a insert and ligation with the backbone vector, and 3) search of new strong promoter. However there are several limitations for these strategies in that it is hard to replicate the DNA, is too large to transformed which causes genetic instability, and is hard to predict promoter strength. In this study, I modified the promoter region and tested on the promoter strength. In addition, I tried to introduce another cis-acting elements such as a transcriptional terminator and RBS (ribosome binding site) to improve the expression of recombinant protein.
Lactococcus lactis subsp. lactis IL1403 is widely used in the dairy and animal industries, and it is also studied for a live oral vaccine product to elicit mucosal immune response. The translational elongation factor Tu (tuf) gene is a house-keeping gene, and tuf promoter is characterized as a strong promoter in IL1403. In this study, tuf promoter was modified to test the efficiency of protein expression using the luciferase gene as a reporter.
Firstly two terminators, TrrnB and TpepN were tested for the luciferase gene expression efficiency. TpepN terminator showed better performance in luciferase expression.
Next, series of tuf promoter modification were attempted. In bacteria, RNA polymerases and several sigma factors recognized and recruited approximately -35 and -10 region upstream from the transcription start site. The core region including -35 and -10 hexamers in tuf promoter (119 bp) was amplified and series of modified tuf promoters were constructed using PCR with partial complementary reverse primer. There PCR products (#1) were cloned into the promoterless pIL.Ptuf.Luc(X) vector. Luciferase activity of t2, t4, t6 and t7 were higher than control tuf promoter. Especially t2 and t4 showed better performance, thus selected for next experiment.
It is well known that the sequence between RBS and start codon (ATG) are important for protein translation efficiency. Thus, I modified original sequence of this region, 'CATTTTTCAT' to 'AATTTTTAAA' to give more AT-rich. This modification was combined with selected modified tuf promoter to give a series of new tuf promoter cassette. The transformed IL1403s containing modified promoter (#2) were assayed for luciferase activity. Derivative of t2 and t4, t2-1 and t4-1 showed better performance.
Combined all the modified clones, luciferase activity was compared. t4-1 showed much higher activity compared to the t4, indicating the sequence between RBS and start codon is important for protein expression. To confirm this results, luciferase expression was analyzed on SDS-PAGE and western blot assay. Luciferase bands (61 kDa) was not detectable in SDS-PAGE, but in western blot, clones with t2, t4, t4-1 showed stronger signal compared to original tuf one.
In conclusion, this study revealed that introduction of modified strong promoter and additional cis-acting elements can improve the protein expression in IL1403. And this strategy has a prospect to improve recombinant protein expression. Since pIL252 is a low copy plasmid-based expression system, high copies-based plasmids are needed to increase recombinant protein expression.Summary I
Contents IV
List of Tables and Figures VII
List of Abbreviations Iโ
ฉ
I. Introduction 1
II. Review of Literature 3
1. Lactic Acid Bacteria (LAB) 3
1) Lactic Acid Bacteria 3
2) LAB as probiotics 4
3) Lactococcus lactis 7
2. Recombinant protein expression 8
1) Plasmid-based expression system 8
2) Recombinant protein expression strategy 8
3. Cis-acting elements 9
1) Promoter 9
2) Cis-acting elements 10
4. The tuf gene 11
III. Materials and Methods 12
1. Bacterial cultivation 12
1) Culture medium 12
2) Cultivation and harvest of bacterial cells 12
2. Transformation of bacteria 13
1) Preparation of IL1403 competent cells 13
2) Transformation of bacterial cells 13
3. DNA works 14
1) Plasmid 14
2) Preparation of plasmid DNA 14
3) Enzyme treatment 17
4) PCR reaction 17
5) PCR purification 17
6) Analysis of nucleotide sequences 18
4. Luciferase assay 20
1) Growth phase-dependent luciferase expression 20
2) Detection of chemiluminescence 20
5. Protein works 21
1) Protein extraction from LAB cells 21
2) Quantification of proteins 21
3) SDS-PAGE and western blot assay 21
4) Intensity measurement of protein bands 22
6. In vitro characterization 23
1) Growth of LAB cells 23
2) pH measurement of LAB cultures 23
IV. Results and Discussion 24
1. Introduction of reporter gene with terminator 24
1) Cloning of luciferase gene 24
2) Vector construction 25
2. Validation of reporter gene and terminator 26
1) Growth characteristics 26
2) Luciferase assay 27
3. Modification of tuf promoter (#1) 28
1) Amplification of short fragments 28
2) Construction of repeated fragments 29
3) Elongation of ribosome binding site (RBS) and enzyme sites 30
4) Introduction of modified promoter (#1) 31
4. Modified promoter (#1) activity assay 35
1) Growth characteristics (#1) 35
2) Luciferase assay (#1) 35
5. Construction of remodified promoter (#2) 37
1) Core promoter cassette preparation from modified promoter (#1) 37
2) Elongation of RBS and modified downstream sequences 38
6. Remodified promoter (#2) activity assay 41
1) Luciferase assay (#2) 41
2) Final luciferase assay 42
7. Confirmation of Protein expression 44
1) SDS-PAGE and western blot assay 44
V. Literature Cited 46
VI. Summary in Korean 58
VII. Acknowledgement 61Maste
๋ค๊ณต์ฑ๋ฌผ์ง์์์ ์์ฐ๋๋ฅํ์์ ๋ํ ์ด์ญํ์ ๊ตญ์ํํ์ํ ๊ฐ์ ์ ๊ณ ์ฐฐ
ํ์๋
ผ๋ฌธ(์์ฌ)--์์ธ๋ํ๊ต ๋ํ์ :๊ธฐ๊ณ๊ณตํ๊ณผ,2000.Maste
(The) study of the actual condition of visiting nurse projects in public health centers in Seoul
์ง์ญ์ฌํ ๊ฐํธํ๊ณผ/์์ฌ[ํ๊ธ]
๋ฐฉ๋ฌธ๊ฐํธ์ฌ์
์ ๊ธฐ๋ณธ ์ ์ฑ
๋ฐฉํฅ์ ๊ตญ๋ฏผ ๋๊ตฌ๋ ํฌ๊ด์ ์ธ ๋ณด๊ฑด์๋ฃ์ ํํ์ฑ๊ณผ ๋๋ฑํ ์ ๊ทผ๋๋ฅผ ์ ์งํ์ฌ ์ง์ ์ธ ์ถ์ ์ ์งํ๊ฒ ํ๋ฉฐ ๋ณต์ง์ฌํ๋ฅผ ์คํํ๊ณ ์ ํจ์ด๋ค.
๋ณธ ์ฐ๊ตฌ๋ ๋ณด๊ฑด์ ๋ฐฉ๋ฌธ๊ฐํธ์ฌ์
์ ์กฐ์ง, ์ธ๋ ฅ, ์์ฐ, ์ค๋น๋, ์
๋ฌด๋ฅผ ์ดํด๋ณด๊ณ ์ด๋ฅผ ๊ธฐ์ด๋ก ํจ์จ์ ์ธ ๋ฐฉ๋ฌธ๊ฐํธ์ฌ์
์ ์ ์ํ๊ณ ์ ์๋ํ์๋ค.
์ฐ๊ตฌ๋ฐฉ๋ฒ์ผ๋ก๋ ๋ฐฉ๋ฌธ๊ฐํธ์ฌ์
์ ๋ฐฉํฅ์ค์ ์ ๊ดํ ๋ฌธํ๊ณ ์ฐฐ๊ณผ ๊ตญ๋ด ๊ธฐ๊ด์ ํต๊ณ์๋ฃ๋ฅผ ์ฌ์ฉํ์๊ณ ๋ํ ์์ธ์ 25๊ฐ ๋ณด๊ฑด์ ๋ฐฉ๋ฌธ๊ฐํธ์ฌ์
๋ด๋น์ 221๋ช
์ ๋ชจ์ง๋จ์ผ๋ก ํ์ฌ ์ฐํธ์ ์ด์ฉํ์ฌ ์ค๋ฌธ์กฐ์ฌํ์๋ค. ์๋ฃ์์ง ๊ธฐ๊ฐ์ 1996๋
11์ 2์ผ ๋ถํฐ 1996๋
11์ 16์ผ๊น์ง์์ผ๋ฉฐ ๋ถ์์ ์ฌ์ฉ๋ ์ฐ๊ตฌ๋์์ ์๋ 23๊ฐ ๋ณด๊ฑด์ 184๋ช
์ด์๋ค.
๋ณธ ์ฐ๊ตฌ์ ์ฃผ์ ๊ฒฐ๊ณผ๋ ๋ค์๊ณผ ๊ฐ๋ค.
1. ์กฐ์ง ํํฉ
์์ธ์๋ ์ด 25๊ฐ์ ๋ณด๊ฑด์๊ฐ ์์ผ๋ฉฐ ํฌ๊ฒ ๋๋์ด ์ง์ญ๋ณด๊ฑด๊ณผ๋ฅผ ๋๊ณ ๋
๋ฆฝ์ ์ผ๋ก ๋ฐฉ๋ฌธ๊ฐํธ์ฌ์
์ ๋ด๋นํ๋ 8๊ฐ๊ตฌ ๋ณด๊ฑด์์ ๊ทธ๋ ์ง ์์ 17๊ฐ๊ตฌ ๋ณด๊ฑด์๋ก ๋๋์ด ์ง๋ฉฐ ํ์กด ์กฐ์ง์ผ๋ก๋ ๋ฐฉ๋ฌธ๊ฐํธ์ฌ์
์ ์ํํ๋๋ฐ ํ๊ณ๊ฐ ์๋ค๊ณ ํ ์ ์๋ค.
2. ์ธ๋ ฅํํฉ
์์ธ์ ๋ณด๊ฑด์์ ํ์์ 95๋
ํ์ฌ ์ด 2,020๋ช
์ด๋ฉฐ ์ด์ค ๊ฐํธ์ง์ด 647๋ช
์ผ๋ก 32%๋ฅผ ์ฐจ์งํ๊ณ ์์ผ๋ ๊ทธ ์ง๊ธ์ ์๋์ ์ผ๋ก ๋ฎ์์ ์ ์ ์๋ค.
๋ณธ ๋
ผ๋ฌธ์์ ์กฐ์ฌํ 184๋ช
์ ๋์์ผ๋ก ์๋ต์์ ์ผ๋ฐ์ ํน์ฑ์ ์ดํด ๋ณด๋ฉด ๋ณด๊ฑด๊ด๋ฆฌ์๋ํ๊ท 46.5์ธ, ๋ฐฉ๋ฌธ๊ฐํธ์ฌ๋ ํ๊ท 35.7์ธ๋ก ๋ํ๋ฌ์ผ๋ฉฐ ๊ต์ก์์ค์ ์ ๋ฌธ๋ ์กธ์
์๊ฐ ๊ฐ์ฅ ๋ง์ ๋ถ๋ถ์ ์ฐจ์งํ๊ณ ์์๋ค. ํน๋ณ๊ต์ก๊ณผ์ ์ค CPHN๊ณผ ๊ฐ์ ๊ฐํธ์ฌ ๊ต์ก์ ๋๋ถ๋ถ์ด ์ด์
ํ์ง ์์ ๊ฒ์ผ๋ก ๋ํ๋ฌ๋ค. ์ด๋ค์ ์ง๊ธ์ ๋ณด๊ฑด๊ด๋ฆฌ์๋ 6๊ธ, ๋ฐฉ๋ฌธ๊ฐํธ์ฌ๋ 8๊ธ์ด ๊ฐ์ฅ ๋ง์ ๊ฒ์ผ๋ก ์กฐ์ฌ๋์์ผ๋ฉฐ 5๊ธ ์ด์ ์๋ต์ 6๋ช
์ค 2๋ช
๋ง ๊ฐํธ์ง์ผ๋ก ์กฐ์ฌ๋์ด ์์ฌ๊ฒฐ์ ๊ถ์ด ๋ฏธ์ฝํจ์ ์ ์ ์์๋ค. ํ๋ถ์ ๊ทผ๋ฌด๊ธฐ๊ฐ์ ๋ฐฉ๋ฌธ๊ฐํธ์ฌ์ ๊ฒฝ์ฐ ํ๊ท 1.5๋
์ผ๋ก ์ด๋์ด ์ฆ์์ ์ ์ ์์์ผ๋ฉฐ ์์๊ฒฝ๋ ฅ์ ํ๊ท 5๋
์ด์์ผ๋ก ์ง์ ์๋น์ค์ ์ ๊ณต์ ์ํด ๋ฐ๋์งํ ๊ตฌ์ฑ์ด๋ผ๊ณ ๋ณผ ์ ์๋ค.
3. ์์ฐ ํํฉ
๋ฐฉ๋ฌธ๊ฐํธ์ฌ์ ์์ฐ์ ๋ํ ๋ง์กฑ๋๋ ์ ๋ฐ์ ์ผ๋ก ๋ฎ์ ๊ฒ์ผ๋ก ๋ํ๋ฌ์ผ๋ฉฐ ์ฐจ๋์ง์ ๋ฐ ์์ค ๋ฐ ์ฅ๋น์ ๋ํด์๋ ๋ถ๋ง์กฑํ๋ค๋ ์๋ต์ด ๋์๋ค. ๋ํ ์์ฐํธ์ฑ์์ ๋ณด๊ฑด๊ด๋ฆฌ์๋ ์ ์ถ์ฑ ์๊ฒ ์์ฐ ํธ์ฑ์ด ์ด๋ฃจ์ด์ง๋ค๊ณ ์๋ตํ ๋ฐ๋ฉด ๋ฐฉ๋ฌธ๊ฐํธ์ฌ๋ ์์ฐ ํธ์ฑ์ด ์ ์ถ์ ์ด
์ง ๋ชปํ๋ค๊ณ ์๋ตํ์ฌ ์๋ฐ๋ ๊ฒฌํด๋ฅผ ๋ณด์๋ค.
4. ๋ฐฉ๋ฌธ๊ฐํธ์ฌ์
์ ๋ํ ๋ฐฉ๋ฌธ๊ฐํธ์ฌ์ ์ค๋น๋
๋ฐฉ๋ฌธ๊ฐํธ์ฌ์
์ ๋ํ ์ค๋น๋์์๋ ๋ฐฉ๋ฌธ๊ฐํธ์ฌ์
์ ๋ํ ๊ต์ก์ ๊ธฐํ๊ฐ ๋ณด๋ค ํญ ๋๊ฒ ์ด๋ฃจ์ด ์ ธ์ผ ํจ์ ์ ์ ์์์ผ๋ฉฐ ์ด๋ค์ด ์ํ๋ ๊ต์ก๋ฐฉ๋ฒ์ผ๋ก๋ ๋ณด๊ฑด๊ด๋ฆฌ์๋ ์ธ๋ฏธ๋์ ๋จ๊ธฐ์ฐ์๋ฅผ, ๋ฐฉ๋ฌธ๊ฐํธ์ฌ๋ ๋จ๊ธฐ์ฐ์์ ์ ๊ท๋ํ /๋ํ์์ ์ ํธํ๋ ๊ฒ์ผ๋ก ๋ํ๋ฌ๋ค.
5. ์
๋ฌดํํฉ
์์ธํน๋ณ์์ ๋ฐฉ๋ฌธ๊ฐํธ์ฌ์
์ ์ํ๋ณดํธ๋์์์ ์ ์๋ ์ฃผ๋ฏผ์ ๋์์ผ๋ก ํ์๊ด๋ฆฌ, ๊ฑด๊ฐ๊ด๋ฆฌ, ์ฐ๊ณ์ฒ๋ฆฌ, ์ํ์ง๋ฃ, ๋ณด๊ฑด๊ต์ก์ ํ๊ณ ์์๋ค.
๋ฐฉ๋ฌธ๊ฐํธ์ฌ์
์ ์
๋ฌด ํํฉ์ ์ดํด๋ณด๋ฉด ๋น๊ต์ ์ ๋๊ณ ์๋ ๊ฒ์ผ๋ก ๋ํ๋ฌ์ผ๋ฉฐ ํนํ ํ์์ ๋ํ ์ถํ๊ด๋ฆฌ์ ์ฃผ๋ฏผ์ ๋์์ผ๋ก ํ๋ ํน์์ฌ์
์์ ์๋๊ณ ์๋ค๊ณ ์๋ตํ์๋ค. ๊ทธ๋ฌ๋ ๋ฐฉ๋ฌธ๊ฐํธ์ฌ์
์ ๋ํ ์ฃผ๋ฏผ ์ฐธ์ฌ, ํ ๊ธฐ๊ด๊ณผ์ ์ฐ๊ณ๋ง ํ์ฑ, ์ ์ฐํ์์๋ ์ํํ๊ฒ ์ด๋ฃจ์ด์ง๊ณ ์์ง ์๋ค๊ณ ์๋ตํ์ฌ ๊ฐ์ ์ด ์๊ตฌ๋์๋ค.
6. ๋ฐฉ๋ฌธ๊ฐํธ์ฌ์
์ ์ํฅ์ ๋ฏธ์น๋ ์์ธ
1) ์ง์ญ๋ณด๊ฑด๊ณผ์ ์ ๋ฌด์ ํ ์
๋ฌด๋ณํ๊ณผ์ ๊ด๋ จ์ฑ
์ง์ญ๋ณด๊ฑด๊ณผ๋ ์์ธ์๊ฐ ๋ฐฉ๋ฌธ๊ฐํธ์ฌ์
์ ์ค์ํ๊ธฐ ์ํ ์ผํ์ผ๋ก ์กฐ์ง ๊ฐํธํ์ฌ ์ ์ค๋ ๊ณผ๋ก์ ์ง์ญ๋ณด๊ฑด๊ณผ๊ฐ ์๋ ๊ณณ์์ ํ ์
๋ฌด๋ฅผ ๋ ๋ง์ด ๋ณํํ๊ณ ์๋๊ฒ์ผ๋ก ๋ํ๋ฌ๋ค.
2) ๋ณด๊ฑด๊ด๋ฆฌ์์ ๊ด์ฌ๋์ ๋ฐฉ๋ฌธ๊ฐํธ์ฌ์
๊ณผ์ ๊ด๋ จ์ฑ
๋ฐฉ๋ฌธ๊ฐํธ์ฌ์
์ ๋ํ ๋ณด๊ฑด๊ด๋ฆฌ์์ ๊ด์ฌ๋๋ ๋์ ๊ฒ์ผ๋ก ๋ํ๋ฌ๋ค. ๋ํ ๋ณด๊ฑด๊ด๋ฆฌ์์ ๊ด์ฌ๋๊ฐ ๋์์๋ก ์์ฐ์ ๋ํ ๋ง์กฑ์ฌ๋ถ, ๋ฐฉ๋ฌธ๊ฐํธ์ฌ์
์ ๋ํ ํ๊ฐ, ์ฃผ๋ฏผ์ ๋์์ผ๋ก ํ๋ ํน์์ฌ์
์์ ํต๊ณํ์ ์ผ๋ก ์ ์ํ๊ฒ ๊ด๋ จ์ฑ์ด ๋์๋ค.
์ด์๊ณผ ๊ฐ์ ๊ฒฐ๊ณผ์ ๊ธฐ์ดํ์ฌ ๋ค์๊ณผ ๊ฐ์ด ์ ์ธํ๋ค.
์ฒซ์งธ, ๋ฐฉ๋ฌธ๊ฐํธ์ฌ์
์ ๋ณด๊ฑด์ธ๋ ฅ์ ์ ๊ทน์ ์ธ ์ง์ญ์ฌํ ํ๋์ ์ ์ ๋ก ํ๋ ๊ฒ์ด๋ฏ๋ก ๊ธฐ์กด ์ธ๋ ฅ์ ๊ฐ๋ฐํ์ฌ ์ง์ ์ธ ์ฌ์
์ ์ํํ๊ฒ ํ์ฌ์ผ ํ ๊ฒ์ด๋ฉฐ ๊ทผ๋ฌดํ๋ฉด์ ์๊ฒฉ์ ์ทจ๋ํ ์ ์๋ ์ ๋์ ๋ณด์์ด ์ ์ฑ
์์ ๊ณ ๋ ค๋์ด์ผ ํ ๊ฒ์ด๋ค.
๋์งธ, ์ง์ญ์ฌํ ๊ธฐ์ด์ง๋จ, ๊ด๋ จ๋ถ์์์ ํ์กฐ ๋ฐ ํ ๊ธฐ๊ด๊ณผ์ ์ฐ๊ณ๋ง ํ์ฑ, ํ ์ ๋ฌธ์ง์ข
๊ณผ์ ํ๋ ฅ, ํ์์ ์กฐ๊ธฐ๋ฐ๊ฒฌ ๋ฐ ๋์์์ ๋ํ ์ถํ๊ด๋ฆฌ, ์ฃผ๋ฏผ์ ๋ฐฉ๋ฌธ๊ฐํธ์ฌ์
์ฐธ์ฌ ๋ฐ๋ฐฉ๋ฌธ๊ฐํธ์ฌ์
์ ๋ํ ํ๊ฐ, ์ง์ญยท์ธ๊ตฌยท๋ฉด์ ์ ๊ณ ๋ คํ ๋์์ธ๊ตฌ ๋ถ๋ด ๋ฑ์ ๋ฐฉ๋ฌธ๊ฐํธ์ฌ์
์
๋ฌด๋ฅผ ๊ฐ์ ํ์ฌ์ผ ํ ๊ฒ์ด๋ค.
์
์งธ, ์ง์ญ๋ณด๊ฑด๊ณผ๋ฅผ ์ ์คํ์ฌ์ผ ํ ๊ฒ์ด๋ฉฐ ๊ด๋ฆฌ์์ ๊ด์ฌ๋๊ฐ ๋ฐฉ๋ฌธ๊ฐํธ์ฌ์
์ ์ค์ํ๊ฒ์ํฅ์ ๋ฏธ์น๋ฏ๋ก ๋ด๋น ๊ณผ์ฅ๊ธ ์ด์์ ๊ด๋ฆฌ์๋ ๋์ผ ์ง์ข
์ ์ํด์ ์
๋ฌด ์ง์ยท๊ฐ๋
์ ๋ฐ์์ผ ํ ๊ฒ์ด๋ค.
์ด ์ฐ๊ตฌ์์ ์ ์๋ ๊ฒฐ๊ณผ๋ค์ ๋น์ถ์ด ๋ฐฉ๋ฌธ๊ฐํธ์ฌ์
์ ํจ์จ์ ์ผ๋ก ํ๊ธฐ ์ํด์๋ ์ ๋ฌธ์ธ๋ ฅ์ ์์ฑ, ์กฐ์ง์ ์ฌ๊ฐํธ, ์์ฐ์ ํ๋ณด๋ฅผ ํตํ ์์ค ๋ฐ ์ฅ๋น์ ๋ณด์, ์
๋ฌด์ง์นจ๊ฐ๋ฐ ๋ฑ์์ค๋น๊ฐ ํ์ํจ์ ์ ์ ์์์ผ๋ฉฐ ์์ผ๋ก ์ด ์ฐ๊ตฌ๋ ๋ฐฉ๋ฌธ๊ฐํธ์ฌ์
์ ์ ํฉํ ๋ฐฉ์์ ๋ชจ์ํ๋๋ฐ ์ฐธ๊ณ ์๋ฃ๋ก ์ ์๋์์ผ๋ฉด ํ๋ค.
[์๋ฌธ]
This study aims to comprehensively grasp the trend of National health policy on the exploitation of medical and health resources and to find out what implications the current budget policy and its effect would give for setting up and enforcing the health policy hereafter.
For these purposes, I did a diachronic analysis of the budget under the jurisdiction of the Health and Welfare Affairs, which has been invested with the aim of increasing the number of sickbed of private hospitals as well as of strengthening the function of both private hospitals and public health organizations for the recent five years. And I, also, compared the change in the sickbed number of each of medical facilities and its sickbed occupancy rate with the increased sickbed number through the Special financial resources from the national treasury loans and those from the farming and fishing villagers' tax. The results are as follows:
In the first place, the increasing rate of the budget of general account under the jurisdiction of the Health and Welfare Affairs(which is 19.5% in 1996) is higher than that of National budget, and the component ratio of the Special financial resources from the national treasury loans and the farming and fishing villagers' tax to National budget has risen steadily every year. According to the annual change in National financial support for the purpose of exploiting medical and health resources, the budget of general account to improve the facilities of public health organizations and to strengthen their function was abolished in 1994, when the Special financial resources from the farming and fishing villagers' tax were newly-
set up. But it was recompiled again with the view of supporting the Health Centers in some urban areas in 1996. Also, in 1996 and 1997, the budget for building private dementia hospitals was drawn up. The budget of the Special financial resources of the national treasury loans was made up mainly to increase the sickbeds of the private hospitals and in 1997, the another budget items were newly drawn up for the construction and extension of special purpose hospitals and the conversion of mental nursing homes into mental nursing hospitals. The 45.9% of the budget of the Special financial resources from the farming and fishing villagers' tax was assigned to public health organizations and the rest, 54.1%, to private hospitals.
Secondly, in all hospitals, except such as tuberculous, leprous, and dental hospital, the rate of the sickbed number increased by financing to the total increased sickbed number for the last three years is 80.5%. The 31.1% of the former was given out to six large cities and the 33.6% of it was alloted to Seoul, Inchon and Kyoungki Area and Southern Kyoungsang Area. And the annual change in the allotment of the total sickbed number to each of cities and counties shows that there was more serious regional unbalance in 1996(urban area: 93.3%; county area: 6.7%) than in 1993. Moreover, the 82.3% of the sickbed number increased by financing was in urban area and the 17.7% of it was in county area.
Finally, in hospitals and general hospitals, the rate of the sickbed number increased by financing to the increased sickbed number for the last three years in each of cities and Provinces is as follows: 445.7% in Southern Cheonla Province; 321.0% in Northern Kyoungsang Province; 199.7% in Kwangju Extensive City; 196.8% in Northern Chungchong Province. Although they are all over 100%, the 1996 sickbed occupancy rate of these areas was lower than the average. The annual average rate of the whole country and six large cities has diminished. In Southern Kyoungsang Province, Kyoungki Province and Pusan Extensive City, the component rate of the sickbed number of mental hospitals
increased by financing is higher than any other areas. And also the occupancy rate is relatively high in comparison with other hospitals. In Chinese medicine hospitals, the 55.3% of the sickbeds increased by financing was alloted to six large cities and the 43.5% was to Seoul, Inchon and Kyoungki Area. But the 1996 sickbed occupancy rate of this Area is lower than the national average rate. The average sickbed occupancy rate
of Chinese medicine hospitals, compared with that of other kind of hospitals, was reduced to a very high degree.
As we can see in the above results, the project of the Special financial resources from national treasury loans and from the farming and fishing villagers' tax (which was originally intended to achieve the equity of medical services through increasing
the sickbed number of private hospitals and reinforcing the function of both private hospitals and public health organizations), in effect, has resulted in the aggravation
of the regional maldistribution primarily because the project increased the sickbed without considering its efficiency.
Conclusively, to improve the equity of the sickbed number of private hospitals and the efficiency in running the sickbeds of private hospitals, I believe that it's necessary to review the current financial policy critically and to establish the directionality of the future financial policy.restrictio
Vascularized bone grafts based on gelatin-heparin conjugated cryogels
ํ์๋
ผ๋ฌธ (์์ฌ)-- ์์ธ๋ํ๊ต ๋ํ์ : ๊ณต๊ณผ๋ํ ํํ์๋ฌผ๊ณตํ๋ถ, 2018. 2. Nathaniel Hwang.A cryogel is a one of the scaffolds which have high porosity with interconnected macropores providing cell compatible microenvironment. In addition, cryogels can be utilized in minimally invasive surgery due to its sponge-like properties, including rapid shape recovery and injectability. Herein, we developed an injectable cryogel by conjugating heparin to gelatin as a vascular endothelial growth factor (VEGF) and fibroblasts carrier for the treatment of ischemic hind limb mouse model. Our gelatin/heparin cryogel showed a mesh-like network dependent on the gelatin concentration, having a different mechanical properties in swelling ratio, interconnected porosity and elasticity. In addition, integrating heparin into cryogel allowed controlled release of VEGF for a long period, leading to a therapeutic effect demonstrated in in vitro endothelial cell angiogenesis study. Also, its sponge-like properties enabled cryogels to be applied as an injectable system with desirable maintenance of cells and proteins after injection. And, as gelatin is one of the natural polymers that have a high biocompatibility, gelatin/heparin cryogel showed high cell loading efficiency and viability without any other treatment. As a result, overall in this study, we optimized the combination of the concentration of gelatin and heparin in aspect of sustainable release of VEGF and injectability. Finally, optimized gelatin/heparin cryogel integrated with VEGF and NIH-3T3 fibroblasts was applied to in vivo ischemic hind limb model and demonstrated its angiogenic potential by improving neovascularization into the cryogel. As utilizing our gelatin/heparin cryogels with human mesenchymal stem cell (hMSCs), we expect to see the therapeutic effect for bone remodeling.1. Introduction 1
2. Experimental section 3
2.1 Preparation of gelatin-heparin cryogels 3
2.2 Characterization of gelatin-heparin cryogels 4
2.3 Rheology test 5
2.4 Heparin detection via alcian blue assay 6
2.5 Synthesis of RITC-heparin conjugated cryogel 6
2.6 Heparin mediated protein release kinetics 7
2.7 HUVEC migration and tube formation assay 7
2.8 Injecatability test 8
2.9 Preparation of cell seeded scaffolds 9
2.10 PicoGreen and live/dead assay 9
2.11 In vivo ischemic hinlimb mouse model 10
2.12 OCT embedding 10
2.13 Histology & immunostaining 11
3. Results and discussion 13
3.1 Fabrication of gelatin/heparin cryogel 13
3.2 Characterization of gelatin/heparin cryogels 14
3.3 Rheological properties of gelatin/heparin cryogels 16
3.4 Heparin conjugated microarchitecture of gelatin/heparin cryogels according to the heparin concentration 17
3.5 Heparin mediated VEGF release kinetics of gelatin/heparin cryogels according to the heparin concentration 18
3.6 In vitro angiogenic response of HUVEC on VEGF dependent on heparin concentration of gelatin/heparin cryogels 20
3.7 Injectability of gelatin/heparin cryogels 21
3.8 Injectable gelatin/heparin cryogel as an effective carrier of NIH-3T3 fibroblasts 23
3.9 Effect of gelatin/heparin cryogels containing VEGF or/and NIH-3T3 fibroblasts on in vivo ischemic hind limb mouse model for neovascularization 24
4. Conclusion 28
REFERENCES 39
์์ฝ (๊ตญ๋ฌธ์ด๋ก) 47Maste