• Title/Summary/Keyword: laser stimulation

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Nuclear and Microtubule Reorganization in Cattle Nuclear Transfered Embryos

  • Shin, Mi-Ra;Park, Sang-Wook;Cui, Xiang-Shun;Shim, Ho-Sup;Kim, Nam-Hyung
    • Proceedings of the KSAR Conference
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    • 2001.03a
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    • pp.18-18
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    • 2001
  • Despite of importance of integrated events of nucleus and microtubule remodeling in nuclear transferred embryos with somatic cells, little information is available on this subject. In this study we configured chromatin and microtubule organization following somatic cell nuclear transfer in pre- and non-activated bovine oocytes in order to clearify nuclear remodeling process and to demonstrate centrosome inheritance during nuclear transfer. The cumulus-oocyte complexes were collected from slaughterhouse and were matured in vitro for 20 h in TCM 199 supplemented hormone. Matured bovine oocytes were enucleated by aspirating the frist polar body and metaphase chromatin using a beveled pipette. Bovine fibroblast cells were fused into enucleated oocyte by electrical stimulation. Reconstructed oocytes were activated with ionomycine and 6-dimethylaminopurin, and then cultured in CRlaa medium. The organization of nuclear and microtubules were observed using laser-scanning confocal microscopy. At 1 hour after fusion, microtubule aster was seen near the transferred nucleus in most oocytes regardless activation condition. While most of fibroblast nuclei remodeled to premature chromosome condensation (PCC) and to the two masses of chromosome in non-activated oocytes, a few number of fibloblasts went to PCC and multiple pronuclear like structures in activated oocytes. Microtubular spindle was seen around condensed chromosome. Gamma-tubulin was detected in the vicinity of condensed chromosome, suggesting this is a transient spindle. The spindle seperated nucleus into two masses of chromatin which developed to the pronuclear like structures. Two pronuclear like structures were than apposed by microtubular aster and formed one syngamy like nuclear structure at 15 h following nuclear transfer. At 17 to 18 h after fusion, two centrosomes were seen near the nucleus, which nucleates micrtubules for two cell cleavage. While 31% of reconstructed oocytes in non-activated condition developed to morulae and blastocysts, a few reconstructed oocytes in pre-activated condition developed to the blastocyst. These results suggested introduction of foreign centrosome during nuclear transfer, which appeared to give an important role for somatic cell nuclear reprogramming.

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Microtubule Assemblies and Methylation Patterns of Porcine IVF and Parthenogenetic Embryos (돼지 체외수정란 및 단위발생란의 미세관 형성 및 메틸화 양상)

  • Park, Joo-Hee;Kim, Ho-Jeong;Lee, Beom-Ki;Kwon, Dae-Jin;Hwang, In-Sun;Park, Choon-Keun;Yang, Boo-Keun;Cheong, Hee-Tae
    • Reproductive and Developmental Biology
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    • v.33 no.1
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    • pp.7-11
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    • 2009
  • This study was performed to confirm the microtubule assemblies and methylation patterns of porcine IVF and parthenogenetic embryos. Cumulus-oocyte complexes were collected and matured in vitro for 42 hr. Oocytes were fertilized by prepared fresh sperm or activated parthenogenetically by exposure to electric stimulation and 6-dimethylaminopurine. Porcine IVF and parthenogenetic embryos were cultured in vitro for 6 days. Embryos were stained by immunofluorescence staining method to observe the dynamic of nucleus and microtubules in the first mitotic phase and the methylation patterns in different developmental stages. After then, samples were confirmed and analyzed through a laser-scanning confocal microscope. IVF embryos had a centrosome originated from sperms, which was shown a $\gamma$-tubulin spot. However, $\gamma$-tubulin spot was not observed in parthenogenetic embryos. A lower methylation level was observed in IVF embryos compared to parthenogenetic ones at the morula and blastocyst stages. In conclusion, it is considered that microtubule assemblies and genetic regulation mechanism differ between parthenogenetic and IVF embryos.

Proteomic Analysis of Rat PC12 Cells Exposed to Cyclosporin A

  • Jung, Ji-Yeon;Seol, Kwang;Jeong, Yeon-Jin;Kim, Won-Jae;Oh, Sang-Jin
    • International Journal of Oral Biology
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    • v.34 no.1
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    • pp.29-36
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    • 2009
  • Cyclosporin A (CsA) has been used clinically as an immunosuppressive drug to prevent organ transplant rejection and in basic research as a mitochondrial permeability blocker. It has been reported that CsA has a protective role in severed neurons and a neurotrophic effect in neuronal cells. However, the molecular mechanisms underlying the stimulation of neuronal cell proliferation by CsA have not yet been elucidated. In our current study, we investigated CsA responsive proteins in PC12 cells using a systematic proteomic approach. The viability of these cells following CsA treatment increased in a dose- and time-dependent manner. Proteins in the CsA-treated PC12 cells were profiled by two-dimensional gel electrophoresis (2-DE) and identified by matrix-assisted laser desorption ionization time-of flight (MALDI-TOF) and electrospray ionization quadupole time-of-flight mass spectrometries (EIQ-TOFMS). This differential expression analysis showed significant changes for 10 proteins (6 up-regulated and 4 down-regulated) upon CsA treatment that were related to cell proliferation, metabolism and the stress response. These proteomics data further our understanding of the proliferation mechanisms of PC12 cells exposed to CsA and demonstrate that our methodology has potential to further elucidate the mechanisms and pathways involved.

ER71/ETV2 Promotes Hair Regeneration from Chemotherapeutic Drug-Induced Hair Loss by Enhancing Angiogenesis

  • Lee, Tae-Jin;Kang, Hee-Kyoung;Berry, Jeffrey C.;Joo, Hong-Gu;Park, Changwon;Miller, Mark J.;Choi, Kyunghee
    • Biomolecules & Therapeutics
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    • v.29 no.5
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    • pp.545-550
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    • 2021
  • Chemotherapy-induced alopecia and hair loss can be stressful in patients with cancer. The hair grows back, but sometimes the hair tends to stay thin. Therefore, understanding mechanisms regulating hair regeneration may improve the management of chemotherapy-induced alopecia. Previous studies have revealed that chemotherapeutic agents induce a hair follicle vascular injury. As hair growth is associated with micro-vessel regeneration, we postulated that the stimulation of angiogenesis might enhance hair regeneration. In particular, mice treated with 5-fluorouracil (5-FU) showed delayed anagen initiation and reduced capillary density when compared with untreated controls, suggesting that the retardation of anagen initiation by 5-FU treatment may be attributed to the loss of perifollicular micro-vessels. We investigated whether the ETS transcription factor ETV2 (aka ER71), critical for vascular development and regeneration, can promote angiogenesis and hair regrowth in a 5-FU-induced alopecia mouse model. Tie2-Cre; Etv2 conditional knockout (CKO) mice, which lack Etv2 in endothelial cells, presented similar hair regrowth rates as the control mice after depilation. Following 5-FU treatment, Tie2-Cre; Etv2 CKO mice revealed a significant reduction in capillary density, anagen induction, and hair restoration when compared with controls. Mice receiving lentiviral Etv2 injection after 5-FU treatment showed significantly improved anagen induction and hair regrowth. Two-photon laser scanning microscopy revealed that enforced Etv2 expression restored normal vessel morphology after 5-FU mediated vessel injury. Our data suggest that vessel regeneration strategies may improve hair regrowth after chemotherapeutic treatment.

Effect of Fusion Condition on In Vitro Development of Caprine Cloned Oocytes with Nuclear Transfer (재래산양에 있어서 핵이식란의 융합조건이 융합 및 체외발달에 미치는 영향)

  • 박희성;김태숙;이윤희;정수영;이명열;홍승표;박준규;김충희;정장용
    • Reproductive and Developmental Biology
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    • v.28 no.2
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    • pp.127-132
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    • 2004
  • This study was conducted to examine the effects of electric stimulation conditions on in vitro developmental ability of caprine embryos after somatic cell nuclear transfer. Recipient oocytes were surgically collected after superovulation by using CIDR and FSH, PMSG, hCG and estrous synchronization in Korean native goats. The caprine ear cells were cultured in vitro in serum-starvation condition (TCM-l99 + 0.5% FBS) for 3 to 5 days of cell confluence. The zona pellucida of in vivo and in vitro matured oocytes were partially drilled using laser system. Single somatic cell was individually transferred into the enucleated oocyte. The reconstructed oocytes were electrically fused with 0.3M mannitol. After the electofusion, embryos were activated by electric stimulation or Ionomycin + 6-DMAP. Nuclear transfer embryos were cultured in mSOF medium supplemented with 0.8% BSA 6∼7 days at 39 , 5% $CO_2$, 5% $O_2$, 90% $N_2$. The fusion rate of donor cells was 60.4% and 40.3 % in ear cell and fetal fibroblast, and cleavage rate were 40.6% and 48.2%, respectively. No significant difference was found in the fusion and cleavage rate in different donor cells. Nuclear transferred oocytes were fused by electric pulses of 1.30∼1.40, 2.30∼2.39 and 2.40∼2.46 ㎸/cm. There was no significant difference among different electric pulses in fusion rates (26.7, 34.8 and 43.8%). The cleavage rate was higher (p<0.05) in 1.30∼1.40 ㎸/cm (82.9%) than 2.30∼2.39 ㎸/cm (43.8%) and 2.40∼2.46 ㎸/cm. (51.8%). The fusion rates of recipient oocyte source were 1st (43.5% and 23.6%), 2nd (55.7% and 39.2%) and 3rd (66.1% and 52.8%) in in vivo and in vitro oocytes. However, fusion ratee were significantly higher (p<0.05) in in vivo than in vitro oocyte. The cleavage rate of fused oocytes from in vivo and in vitro sources were 52.6% and 54.4%, respectively. No significant difference was found in the cleavage rate according to the recipient oocyte source. These results suggest that factors such as field pulse of electric stimulation and oocyte source could affect in vitro developmental ability of nuclear transplanted caprine oocytes.

Comparative study of acupuncture and invasive laser acupuncture therapy at $SI_3$.$BL_{40}$ on the tibial, sural nerve injury and L5 spinal nerve ligation model in rats (백서(白鼠)의 신경병리성(神經病理性) 동통(疼痛)에 대한 후계(後谿).위중(委中) 혈위(穴位) 호침료법(毫鍼療法)과 레이저 침습조사(侵襲照射) 침료법(鍼療法)과의 비교(比較) 연구(硏究))

  • Wei, Tung-Sheun;Youn, Dae-Hwan;Youn, Yeo-Chung;Na, Chang-Su
    • Korean Journal of Acupuncture
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    • v.22 no.2
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    • pp.9-24
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    • 2005
  • Objective: We have studied the effects of acupuncture and low level He-Ne laser therapy(LLLT) at $SI_3$, $BL_{40}$ on the tibial, sural nerve injury due to sports-damage or traffic accident and L5 spinal nerve ligature model like general herniation of nucleus pulposus(HNP) in a rat of neuopathic pain. Methods: A model of neuropathic pain was made by injuring tibial nerve and sural nerve while common peroneal nerve was maintained. Also, it was made by isolating left 5th lumbar spinal nerve. Three weeks after the neuropathic surgery, acupuncture and LLLT was injected at $SI_3$,$BL_{40}$ one time a day for one week. LLLT was divided three groups, that is LLLT-1(5mW), LLLT-2(10mW) and LLLT-3(30mW). After that, we examined the withdrawal response of neuropathic rats' legs by Von frey filament and acetone stimulation. And also we examined c-Fos, Nocieptin and KOR-3 in the midbrain central gray of neuropathic rats. Results: As we have observed the effect of mechanical allodynia, LLLT-3 group were diminished on 4 day, 5 day, 6 day and 7 day in the resection model compared with control model, LLLT-1 group were diminished on 5 day, LLLT-2 group were diminished on 3 day and 6 day, LLLT-3 group were diminished on 3 day, 4 day, 5 day, 6 day and 7 day in connected model compared with control group. As we have observed the effect of cold allodynia, LLLT-3 group were diminished on 7 day in the resection model compared with control model, LLLT-1 group were diminished on 6 day, 7 day, LLLT-3 group were diminished on 7 day in connected model compared with control group. As we have observed the effect of activity of c-Fos in the central gray part, LLLT-3 were diminished in resection model compared with control group, LLLT-1 group were diminished in connected model compared with control group. As we have observed the effect of activity of Nociceptin in the central gray part, resection model were not increased compared with control group, LLLT-1 group and LLLT-3 group were increased in connected model compared with control model. As we have observed the effect of activity of KOR-3 in the central gray part, resection model were not increased compared with control group, LLLT-3 group were increased in connected model compared with control model. Conclusions: We have noticed that LLLT-1 and LLLT-3 group have more controllable effect than acupuncture group. This study can be used in clinical therapy for neuropathic pain. But it is not reliability that Nociceptin and KOR-3 have effectively to control pain. Therefore We have to follow up about that.

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Response of osteoblast-like cells cultured on zirconia to bone morphogenetic protein-2

  • Han, Seung-Hee;Kim, Kyoung-Hwa;Han, Jung-Seok;Koo, Ki-Tae;Kim, Tae-Il;Seol, Yang-Jo;Lee, Yong-Moo;Ku, Young;Rhyu, In-Chul
    • Journal of Periodontal and Implant Science
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    • v.41 no.5
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    • pp.227-233
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    • 2011
  • Purpose: The aim of this study was to compare osteoblast behavior on zirconia and titanium under conditions cultured with bone morphogenetic protein-2. Methods: MC3T3-E1 cells were cultured on sandblasted zirconia and sandblasted/etched titanium discs. At 24 hours after seeding MC3T3-E1, the demineralized bone matrix (DBM) gel alone and the DBM gel with bone morphogenetic protein-2 (BMP-2) were added to the culture medium. The surface topography was examined by confocal laser scanning microscopy. Cellular proliferation was measured at 1, 4, and 7 days after gel loading. Alkaline phosphatase activity was measured at 7 days after gel loading. The mRNA expression of ALPase, bone sialoprotein, type I collagen, runt-related transcription factor 2 (Runx-2), osteocalcin, and osterix were evaluated by real-time polymerase chain reaction at 4 days and 7 days. Results: At 1, 4, and 7 days after loading the DBM gel alone and the DBM gel with BMP-2, cellular proliferation on the zirconia and titanium discs was similar and that of the groups cultured with the DBM gel alone and the DBM gel with BMP-2 was not significantly different, except for titanium with BMP-2 gel. ALPase activity was higher in the cells cultured with BMP-2 than in the other groups, but there was no difference between the zirconia and titanium. In ALPase, bone sialoprotein, osteocalcin, Runx-2 and osterix gene expression, that of cells on zirconia or titanium with BMP-2 gel was much more highly increased than titanium without gel at day 7. The gene expression level of cells cultured on zirconia with BMP-2 was higher than that on titanium with BMP-2 at day 7. Conclusions: The data in this study demonstrate that the osteoblastic cell attachment and proliferation of zirconia were comparable to those of titanium. With the stimulation of BMP-2, zirconia has a more pronounced effect on the proliferation and differentiation of the osteoblastic cells compared with titanium.

The Analysis of Research Trend about Management of Low Back Pain (요통관리에 관한 연구동향 분석)

  • Hyun, Kyung-Sun
    • The Korean Journal of Rehabilitation Nursing
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    • v.1 no.1
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    • pp.51-60
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    • 1998
  • The result of studying papers about management of lower back pain published in domestic and foreign nursing and medical magazines in these 10 years is as follows ; 1. General characteristic of lower back pain ; 1) In sex distribution, there were more men than women in 5 papers and more women than men in 4 papers among 9 papers surveryed. 2) In age distribution, thirties to forties of patients had more lower back pain as surveyed by general hospitals and fifties to sixties of patients had more lower back pain as surveyed by oriental medical hospitals. 3) In cause factor, there were 50 to 65% of sprain, 32 to 44% of herniated intervertebral disc and 13 to 29% of degenerative changes. 4) In symtom distribution, there were 26 to 57% of lower back pain, 42 to 65% of lower back pain with radiating pain and 34 to 99% of paravertevral muscle spasm. 5) In period of pain management distribution, 18 to 40% of patients experienced pain for less than 6 months and 59 to 82% of them experienced pain for more than 6 months in 3 papers among 4 papers. 6) In surveying the treatment, 66 to 88% of patients had conservative treatment and there were treatments of general hospital, oriental medicine, self remedy and traditional practice in conservative treatment. 7) In job distribution, 12 to 50% of them were housekeepers, 23 to 31% office workers, 4.6 to 36% blue color workers and 11 to 15% students. 2. As psychological character lower back pain paients had anxiety, depression, anger-hostility, phobic anxiety, neurasthenia, hypochondriasis, and interpersonal sensitivity. 3. To distinguish the cause of lower back pain, plain lumbar roentgenogram, straight leg rasing test, eletromyelogic findings, somatosensory evoked potentials CT and MRI were performed. 4. To relieve lower back pain. epidural adhesiolysis, epidulal injection of local anesthetic in mixture with steroid, lumbar spinal root block, low level laser therapy, acupuncture like transcutaneous nerve stimulation(AL TENS), topical capsaicin and lumbar orthotics were used in medical field, and relaxation technique was used in nursing field. 5. Mckenzie's extension exercise and William's flexsion exercise for lower back pain were used in medical field and Yoga exercise was applied in nursing field. 6. The more school education and self efficacy were high, the better they had active coping lower back pain positively and the less self efficacy was the more they had serious pain. As a result of studying the paper there have been very little research for lower back pain in nursing fields of Korea and foreign countries. Because 60 to 80% of population expeience lower back pain at least more than once, it is necessary to develop the study and clinical practice for management of lower back pain.

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$In$ $Vitro$ Development of Goat Parthenogenetic Oocytes Derived from Different Activation Methods (도축장 유래 산양난자의 단위 발생 유기 방법에 따른 체외 발달)

  • Yun, Yun-Jin;Park, Kyeong-Jin;Park, Hee-Sung
    • Journal of Embryo Transfer
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    • v.27 no.1
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    • pp.57-62
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    • 2012
  • Efficient oocyte activation is a key step for the success of nuclear transfer in cloning. Ionomycin sequentially combined with 6-DMAP is now widely used to activate normal oocytes for analytical studies of oocyte activation and to activate reconstructed oocytes after nuclear transfer. The present study investigated sources of oocytes, duration of ionomycin and 6-DMAP, laser and electric stimulation in goat oocyte activation in order to optimize the protocols. Goat ovaries were collected in individual abattoirs during the breeding season and were delivered to the laboratory within 6 h in saline with 100 IU/ml streptomycin and 0.05 mg/ml penicillin. The oocytes were denuded from the cumulus cell by pipetting with 0.2% hyaluronidase in PBS at 20~22 hr post maturation. Oocytes with the polar body were selected and assigned to four groups for parthenogenetic activation. To examine the effect of duration of ionomycin treatment, oocytes after 20~22 hr of maturation were treated with 2.5 uM ionomycin for 1 or 5 min times and then cultured in 2 mM 6-DMAP for 2 or 4 hr. The activated oocytes were cultured in mSOF at $38.5^{\circ}C$ in $CO_2$ 5%, $O_2$ 5% and $N_2$ 90% multi incubator. Cleavage and blastocyst development was observed at 48 hr and day 8 of culture $in$ $vitro$, respectively. Activation rates of oocytes exposed to ionomycin for 1 min(86.4%) were significantly higher than those treated for 5 min(74.3%) duration. This indicated that 1 min ionomycin treatment was most suitable for activation of goat oocytes. The duration of 6-DMAP treat duration was in 2 mM 6-DMAP for 2 hr after 1 min exposure to 2.5 uM ionomycin. The activation rate of oocytes incubated in 6-DMAP for 2 hour(82.5%) was significantly higher than those in oocytes treated with 4 hr(75.5%).

Blood Flow Changes in the Masseter Muscle and Overlying Skin Following Various Functional Waves of Transcutaneous Electrical Nerve Stimulation (경피신경자극치료기(TENS)의 기능별 주파수에 따른 교근과 피개상피의 혈류변화에 관한 연구)

  • Cho, Sung-Guk;Auh, Q-Schick;Chun, Yang-Hyun;Hong, Jung-Pyo
    • Journal of Oral Medicine and Pain
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    • v.34 no.1
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    • pp.115-122
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    • 2009
  • The following results were obtained, after experimenting on change of masseter muscle and bloodstream epithelium with Transcutaneous Electrical Nerve Stimulator(TENS), among 16 male adults. 1. According to applying TENS, it was observed that bloodstream in muscle increases at 1.5, 3.0 Hz. 2. According to applying TENS, it was observed that concentration of moving blood cells in muscle increase at 1.5, 3.0, 6.0 Hz. 3. According to applying TENS, it was observed that velocity of bloodstream in muscle increases only at 1.5 Hz 4. Through experiment, applying TENS at level of 6.0 and 10.0 Hz, all bloodstream, concentration of moving blood cells, and velocity of bloodstream increasing rates were lower in muscle compared to of them in overlying epithelium; and especially increasing bloodstream and its velocity were most frequent at 6.0 Hz, and bloodstream of 10.0 Hz. From the results above when a physical therapy of TENS is carried out the frequency of 1.5, 3.0 H is effective, and as the frequency increases it is disadvantageous to the muscle.