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백서 장관골의 단백질분해 효소 및 이에 대한 hydrocortisone의 효과

Title
 백서 장관골의 단백질분해 효소 및 이에 대한 hydrocortisone의 효과
Other Titles
 The proteolytic enzyme activity and hydrocortisone effect on protein degradation in long bone of albino rats
Issue Date
1973
Publisher
 연세대학교 대학원
Description
의학과/박사
Abstract
[한글] The Proteolytic Enzyme Activity and Hydrocortisone Effect on Protein Degradation in Long Bone of Albino Rata Chang Do Choi, M.D. Department of Medical Science, The Graduate School, Yonsei University (Directed by Prof. In Hee Chung and Je Hyun Kim) Synthesis of collagen and mucopolysaccharide in the bone is inhibited by glucocorticoid hormone (Layton,1951 ; Schiller and Dorfman, 1957; McCluskey and Thomas, 1959; Bernick and Ershoff, 1963), and triiodothyronine inhibits the incorporation of proline C**l4 into collagen (Halme et al., 1972). Heller-Steinberg (1951) found that osteoclasts produce an enzyme which might be involved in the protein degradation of bone matrix. The administration of excess vitamin A increased the protein degradation (Fell and Mellanby, 1952) and also increased the release of acid protease (Fell and Dingle, 1963 ; Lucy et al., 1961). Ali (1964) suggested that another enzyme may be present in bone matrix for degrading chondroprotein, even though collagenase and hyaluronidase are absent. Pita et al. (1970) reported that the protein degradation process preceded mineral formation. Recently, Halme et al. (1972) observed that triiodothyronine increased the protein degradation in cultured bone. As a whole, these studies were not on proteolytic enzymes, but on degradation phenomena of protein. Only a few proteolytic enzymes in bone matrix have been identified. The purpose of the present study was to investigate proteolytic enzymes in the long bone of albino rat at different stages of the growth and the effect of hydrocortisone on protein degradation. Material and Method a. Adult rats: healthy, female adult rats, weighing around 200 g, were used for obtaining fetuses and newborns. The proestrus stage was checked by vaginal smear and next day the conception was confirmed by the presence of the sperms. b. The fetus was obtained on the 18th day of gestation by laparatomy. c. Rats in growing stages: -rats in the first day and third day of the neonatal period were obtained from our laboratory, and those weighing 40, 100, 150 and 200 g of body weight were purchased from a local market. The culture was carried out according to the method of Raisz(1965). The bone(tibia) was excised under stereomicroscope in the tissue culture room and was cultured in microculture slides or dishes containing BGJb media. Culture was performed in the incubator at 37℃ under 5% CO^^2 for 4 or 6 days, depending on the experimental design, and the media was changed every two days. A preliminary stuffy was made on the growth pattern of tibia and the release of Ca**45 as a measure of evaluation of this culture system. The sliced bone (from 18th day fetus, 1st day and 3rd day infant rats) or the homogenated bone marrow (from rats over 40g) of the tibia was used for measuring cathepsin A, B, C and D activity by the method of Misaka and Tappel (1971). The tibiae of 18th day fetuses and 1 day newborns were incubated for 24 hours in BGJb media with 0.012-0.02 μCi of glutamate-C**l4 (specific activity : 14.9 mCi/mM) in order to incorporate the glutamate-C**l4 into the bony tissue. These cultured bones were divided into 2 groups; one group of bones boiled in water for 10 minutes and the other group of non-boiled bones. Thereafter, bone culture was continued to examine the release of C**l4 into the medium from that incorporated in the bone. After 48 hours of culture, the bones were homogenized and treated with 10% trichloracetic acid, and the radioactivity of C**l4 in the insoluble part and in the soluble part was measured. The radioactivity of C**14 in the media was measured without treatment by 10% trichloracetic acid. Effect of hydrocortisone on protein degradation: The bones were cultured for 6 days in BGJb media with 0.5 mg of hydrocortisone and measured for radioactivity of C**l4 . In the control group, 0.85% saline was added to the media in place of hydrocortisone. Ten adult male rats weighing around 200 g were divided into 2 groups; The control group was injected hypodermically daily for 3 weeds, with 0.5 ml of 0.85% saline and the treated group, with 0.5 ml contained of 6 mg of hydrocortisone for each rat. At the completion of the treatment, the rats were sacrificed by guillotine and measured the activities of cathepsin A,B,C and D in the bone. Also for 3 days prior to sacrifice, urine was collected and hydioxyproline excretion was determined by the method of Neuman and Logan (1950). The radioactivity of C**l4 and Ca**45 was measured by a Packard-Tricab Liquid Scintillation Spectrometer. Results A. Protein degradation in the bone 1. Growth of cultured bone The length of tibia of the 18th day fetus was 1.98±0.05 mm on the average and the growth rate was 0.5 mm after 24-hour culture. 2. Proteolytic enzyme in the bone The activity of cathepsin A, B and D in the growing stages was generally high, and the maximum value was observed in the 100∼150 g group. Cathepsin A was 36.14±2.82 in 40g, 50.05±16.20 in 100g, 46.66±8.16 in 150g and 35.00±1.06 nmol/min/mg of protein in 200 g rats respectively. Cathepsin B was 24.29±11.50 in 40g, 28.57±13.43 in 100g, 26.43±8.29 in 150g and 12.86±6.21 nmol/min/mg of protein in 200g rats. Cathepsin C was 4.50±1.16 in 40g, 6.03±2.22 in 100g, 6.67±1.70 in 150g and 2.51±0.33 nmol/min/mg of protein in 200g rats. Cathepsin D was 13.40±0.13 in 40g, 15.18±1.97 in 100g, 22.55±3.55 in 150 g and 14.20±1.06 nmol/min/mg of protein in 200g rats. 3. Release of glutamate-C**l4 In the 18th day fetus, the radioactivity of C**l4 in the insoluble part of the bone after 4days culture was 243±37.71 cpm in the boiled bone and 218±30.99 cam in the non-boiled bone. The released radioactivity in the media was 107±48.52 cpm in the boiled bone and 201±79.73 cpm in the non-boiled bone. The ratio of the radioactivity of the bone and the media was 1:0.44 in the boiled bone and 1:0.91 in the non-boiled bone. In 1 day old newborn rat, the radioactivity was 1,37l cam in the bone and 559 cpm in the media of boiled group, an4 960 cam in the bone and 1,556 cam in the media of the non-boiled group. The ratio of the radioactivity of the bone and media was 1:0.43 in the boiled bone and 1:1.62 in the non-boiled bone. These data are suggestive of the presence of proteolytic phenomenon in the bone. 4. Hydroxyproline excretion in 24 hours urine was 5.46±0.99, 2.99±0.23, 1.60±0.11 and 0.87±0.17 ㎍ per g of body weight in the 40, 100, 150 and 200g of body weight groups respectively. These observations on the cathepsin activities in the bone and the hydroxyproline excretion in the urine suggest that proteolytic phenomenon in the bone may be mediated by a different proteolytic enzyme in addition to cathepsin. B. Effect of hydrocortisone on protein degradation 1. On proteolytic enzyme in vivo experiment In the hydrocortisone treated group, the activities were 32,40±3.51, 15.70±1.37, 3.00±0.59 and 17.00±2.38 nmol/min/mg of protein for cathepsin A,B,C and D respectively. In the control group, the cathepsin activities of A,B,C and D were 38.80±5.82, 21.10±1.87, 3.00±0.88 and 15.70±2.40 nmol/min/mg of protein respectively. 2. On glutamate-C**l4 release In the 18th day fetus, radioactivity was 218±30.99 cpm in the bone and 201±79.73 cpm. in the media of the control group, and it was 253±24.03 cpm in the bone and 214±77.50 cpm in the media of the hydrocortisone treated group. In 1 day old newborn, it was 960 cpm in the bone and 1,556 cam in the media of the control group, and 740 cam in the bone and 1,331 cpm in the media of the treated group. The ratio of radioactivity of the bone to the media was 1:1.62 in the control group and 1:1.79 in the treated group. 3. On hydroxyproline excretion in urine Twenty four-hour urinary excretion of hydroxyproline was 0.56±0.017 ㎍ per g of body weight in the control group and 0.58±0.044 ㎍ per g of body weight in the hydrocortisone treated group. This result indicates that the hydrocortisone has no significant effects on protein degradation in bone. Conclusion In albino rat bone at different stages of growth, protein degradation and cathepsin A,B,C and D activity were studied in both cultured bone and in vivo experiments. The results are as follows: 1. Proteolytic enzymes, cathepsin A,B,C and D were present in the 18th day fetus, in the 1 and 3 days newborn, and in 40, 100, 150 and 200g group rats. 2. The activity of cathepsin A,B,C and D was gradually increased with age. The maximum value was noted in the 100∼150g group and the value was less in the 200g group. 3. In general, the activity of cathepsin A was higher than that of cathepsin D at each of different growing stages. 4. Twenty four-hour urinary excretion of hydroxyproline per gram of body weight was higher in younger rats than older one. 5. Hydrocortisone failed to exert a significant effect on protein degradation and cathepsin activity in the cultured bone and in vivo experiments. 6. Proteolytic phenomena in the bone may be mediated by different proteolytic enzyme in addition to cathepsin.
[영문] Synthesis of collagen and mucopolysaccharide in the bone is inhibited by glucocorticoid hormone (Layton,1951 ; Schiller and Dorfman, 1957; McCluskey and Thomas, 1959; Bernick and Ershoff, 1963), and triiodothyronine inhibits the incorporation of proline C**l4 into collagen (Halme et al., 1972). Heller-Steinberg (1951) found that osteoclasts produce an enzyme which might be involved in the protein degradation of bone matrix. The administration of excess vitamin A increased the protein degradation (Fell and Mellanby, 1952) and also increased the release of acid protease (Fell and Dingle, 1963 ; Lucy et al., 1961). Ali (1964) suggested that another enzyme may be present in bone matrix for degrading chondroprotein, even though collagenase and hyaluronidase are absent. Pita et al. (1970) reported that the protein degradation process preceded mineral formation. Recently, Halme et al. (1972) observed that triiodothyronine increased the protein degradation in cultured bone. As a whole, these studies were not on proteolytic enzymes, but on degradation phenomena of protein. Only a few proteolytic enzymes in bone matrix have been identified. The purpose of the present study was to investigate proteolytic enzymes in the long bone of albino rat at different stages of the growth and the effect of hydrocortisone on protein degradation. Material and Method a. Adult rats: healthy, female adult rats, weighing around 200 g, were used for obtaining fetuses and newborns. The proestrus stage was checked by vaginal smear and next day the conception was confirmed by the presence of the sperms. b. The fetus was obtained on the 18th day of gestation by laparatomy. c. Rats in growing stages: -rats in the first day and third day of the neonatal period were obtained from our laboratory, and those weighing 40, 100, 150 and 200 g of body weight were purchased from a local market. The culture was carried out according to the method of Raisz(1965). The bone(tibia) was excised under stereomicroscope in the tissue culture room and was cultured in microculture slides or dishes containing BGJb media. Culture was performed in the incubator at 37℃ under 5% CO^^2 for 4 or 6 days, depending on the experimental design, and the media was changed every two days. A preliminary stuffy was made on the growth pattern of tibia and the release of Ca**45 as a measure of evaluation of this culture system. The sliced bone (from 18th day fetus, 1st day and 3rd day infant rats) or the homogenated bone marrow (from rats over 40g) of the tibia was used for measuring cathepsin A, B, C and D activity by the method of Misaka and Tappel (1971). The tibiae of 18th day fetuses and 1 day newborns were incubated for 24 hours in BGJb media with 0.012-0.02 μCi of glutamate-C**l4 (specific activity : 14.9 mCi/mM) in order to incorporate the glutamate-C**l4 into the bony tissue. These cultured bones were divided into 2 groups; one group of bones boiled in water for 10 minutes and the other group of non-boiled bones. Thereafter, bone culture was continued to examine the release of C**l4 into the medium from that incorporated in the bone. After 48 hours of culture, the bones were homogenized and treated with 10% trichloracetic acid, and the radioactivity of C**l4 in the insoluble part and in the soluble part was measured. The radioactivity of C**14 in the media was measured without treatment by 10% trichloracetic acid. Effect of hydrocortisone on protein degradation: The bones were cultured for 6 days in BGJb media with 0.5 mg of hydrocortisone and measured for radioactivity of C**l4 . In the control group, 0.85% saline was added to the media in place of hydrocortisone. Ten adult male rats weighing around 200 g were divided into 2 groups; The control group was injected hypodermically daily for 3 weeds, with 0.5 ml of 0.85% saline and the treated group, with 0.5 ml contained of 6 mg of hydrocortisone for each rat. At the completion of the treatment, the rats were sacrificed by guillotine and measured the activities of cathepsin A,B,C and D in the bone. Also for 3 days prior to sacrifice, urine was collected and hydioxyproline excretion was determined by the method of Neuman and Logan (1950). The radioactivity of C**l4 and Ca**45 was measured by a Packard-Tricab Liquid Scintillation Spectrometer. Results A. Protein degradation in the bone 1. Growth of cultured bone The length of tibia of the 18th day fetus was 1.98±0.05 mm on the average and the growth rate was 0.5 mm after 24-hour culture. 2. Proteolytic enzyme in the bone The activity of cathepsin A, B and D in the growing stages was generally high, and the maximum value was observed in the 100∼150 g group. Cathepsin A was 36.14±2.82 in 40g, 50.05±16.20 in 100g, 46.66±8.16 in 150g and 35.00±1.06 nmol/min/mg of protein in 200 g rats respectively. Cathepsin B was 24.29±11.50 in 40g, 28.57±13.43 in 100g, 26.43±8.29 in 150g and 12.86±6.21 nmol/min/mg of protein in 200g rats. Cathepsin C was 4.50±1.16 in 40g, 6.03±2.22 in 100g, 6.67±1.70 in 150g and 2.51±0.33 nmol/min/mg of protein in 200g rats. Cathepsin D was 13.40±0.13 in 40g, 15.18±1.97 in 100g, 22.55±3.55 in 150 g and 14.20±1.06 nmol/min/mg of protein in 200g rats. 3. Release of glutamate-C**l4 In the 18th day fetus, the radioactivity of C**l4 in the insoluble part of the bone after 4days culture was 243±37.71 cpm in the boiled bone and 218±30.99 cam in the non-boiled bone. The released radioactivity in the media was 107±48.52 cpm in the boiled bone and 201±79.73 cpm in the non-boiled bone. The ratio of the radioactivity of the bone and the media was 1:0.44 in the boiled bone and 1:0.91 in the non-boiled bone. In 1 day old newborn rat, the radioactivity was 1,37l cam in the bone and 559 cpm in the media of boiled group, an4 960 cam in the bone and 1,556 cam in the media of the non-boiled group. The ratio of the radioactivity of the bone and media was 1:0.43 in the boiled bone and 1:1.62 in the non-boiled bone. These data are suggestive of the presence of proteolytic phenomenon in the bone. 4. Hydroxyproline excretion in 24 hours urine was 5.46±0.99, 2.99±0.23, 1.60±0.11 and 0.87±0.17 ㎍ per g of body weight in the 40, 100, 150 and 200g of body weight groups respectively. These observations on the cathepsin activities in the bone and the hydroxyproline excretion in the urine suggest that proteolytic phenomenon in the bone may be mediated by a different proteolytic enzyme in addition to cathepsin. B. Effect of hydrocortisone on protein degradation 1. On proteolytic enzyme in vivo experiment In the hydrocortisone treated group, the activities were 32,40±3.51, 15.70±1.37, 3.00±0.59 and 17.00±2.38 nmol/min/mg of protein for cathepsin A,B,C and D respectively. In the control group, the cathepsin activities of A,B,C and D were 38.80±5.82, 21.10±1.87, 3.00±0.88 and 15.70±2.40 nmol/min/mg of protein respectively. 2. On glutamate-C**l4 release In the 18th day fetus, radioactivity was 218±30.99 cpm in the bone and 201±79.73 cpm. in the media of the control group, and it was 253±24.03 cpm in the bone and 214±77.50 cpm in the media of the hydrocortisone treated group. In 1 day old newborn, it was 960 cpm in the bone and 1,556 cam in the media of the control group, and 740 cam in the bone and 1,331 cpm in the media of the treated group. The ratio of radioactivity of the bone to the media was 1:1.62 in the control group and 1:1.79 in the treated group. 3. On hydroxyproline excretion in urine Twenty four-hour urinary excretion of hydroxyproline was 0.56±0.017 ㎍ per g of body weight in the control group and 0.58±0.044 ㎍ per g of body weight in the hydrocortisone treated group. This result indicates that the hydrocortisone has no significant effects on protein degradation in bone. Conclusion In albino rat bone at different stages of growth, protein degradation and cathepsin A,B,C and D activity were studied in both cultured bone and in vivo experiments. The results are as follows: 1. Proteolytic enzymes, cathepsin A,B,C and D were present in the 18th day fetus, in the 1 and 3 days newborn, and in 40, 100, 150 and 200g group rats. 2. The activity of cathepsin A,B,C and D was gradually increased with age. The maximum value was noted in the 100∼150g group and the value was less in the 200g group. 3. In general, the activity of cathepsin A was higher than that of cathepsin D at each of different growing stages. 4. Twenty four-hour urinary excretion of hydroxyproline per gram of body weight was higher in younger rats than older one. 5. Hydrocortisone failed to exert a significant effect on protein degradation and cathepsin activity in the cultured bone and in vivo experiments. 6. Proteolytic phenomena in the bone may be mediated by different proteolytic enzyme in addition to cathepsin.
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http://ir.ymlib.yonsei.ac.kr/handle/22282913/117258
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