흰쥐의 원위콩팥단위내 칼슘결합단백질 (calbindin-D28k)에 관한 면역세포화학적 연구

김영희,김완영,김인범,한승호,오수자,차정호,김진 대한해부학회 1997 Anatomy & Cell Biology Vol.30 No.6

발생중인 흰쥐 콩팥에서 calbindin D28k 양성세포에 관한 면역세포화학적 연구

박은영(Eun-Young Park),이현욱(Hyun-Wook Lee),송현국(Hyun-Kuk Song),황진선(Jin-Sun Hwang),김완영(Wan-Young Kim),김 진(Jin Kim) 대한해부학회 2007 Anatomy & Cell Biology Vol.40 No.1

Calbindin D28k는 칼슘결합단백질(calcium binding protein)의 일종으로 여러 조직에서 발현되고 있으며, 특히 콩팥의 원위콩팥단위 중 일부 세포에 발현하여 칼슘의 재흡수를 조절하는 것으로 알려져 있다. 성체 흰쥐 콩팥에서의 calbindin D28k 발현에 대해서는 이미 보고한 바 있으나 발생 과정중 발현 시기나 그 분화에 대해서는 알려진 바 없다. 발생 중 흰쥐 콩팥의 속질집합관에서 사이세포는 서로 다른 두 가지 기전에 의해 제거되는데, 이러한 제거 기전에 관한 것은 아직까지 알려진바 없으나, calbindin이 아포프토시스로부터 세포를 보호한다는 여러 보고가 있어, 본 연구는 발생 중 흰쥐 콩팥을 대상으로 calbindin D28k 양성 세포의 정확한 발현 시기와 발생과정에 따른 이들의 분포 변화를 알아보고자 하였다. 실험동물로는 흰쥐 임신 16일, 17일, 18일 및 20일 군과 출생 후 1일, 3일, 5일, 7일, 14일 및 21일군 및 성체 콩팥을 사용하였으며 calbindin D28k에 대한 단클론항체와 사이세포를 표지하기 위한 H+-ATPase에 대한 다클론항체로 단독 혹은 이중 면역조직화학법을 시행하였다. Calbindin D28k 면역반응은 임신 17일군의 연결세관과 속질집합관의 몇몇 세포에서 처음 발현되었다. 출생 전 콩팥의 연결세관에서는 연결세관세포만이 calbindin D28k에 강한 양성반응을 보였고, 연결세관 내 사이세포에서는 어떤 면역반응성도 관찰할 수 없었다. 그러나 속질집합관의 경우 calbindin D28k 양성 세포의 수는 임신 18일부터 현저히 증가하였는데, 양성세포의 대부분은 A형 사이세포였고 B형 사이세포나 주세포는 calbindin D28k 면역반응성이 약화되거나 거의 없었다. 속속질 내 calbindin D28k에 양성반응을 띠는 A형 및 B형 사이세포는 출생 후 14일 동안 콩팥유두의 끝에서부터 점차적으로 사라졌으며, 출생 14일 이후 속속질집합관 및 바깥속질집합관 내 사이세포, 특히 A형 사이세포에서의 calbindin D28k 면역반응은 현저히 감소하였다. 또한 출생 후 14일군의 겉질집합관에서의 calbindin D28k 면역반응은 다소 증가하여 일부 A형 사이세포에서도 관찰되었으며, 출생 후 21일 이후에는 성체와 같은 양상을 보였다. 이상의 결과로 보아, 발생중인 흰쥐 콩팥에서 A형 사이세포와 B형 사이세포내 calbindin D28k의 발현이 서로 다름을 알 수 있었고, 이러한 calbindin D28k의 서로 다른 발현양상은 두 세포의 죽는 양상과 밀접한 관련이 있을 것으로 생각된다. Calbindin D28k, a calcium binding protein, is found in various tissues, including some cells in the distal nephron. It plays an important role in the regulation of calcium reabsorption. We previously reported the expression of calbindin D28k in adult rat kidney. However, the exact time of expression during differentiation in the embryonic kidney is not known. During development, intercalated cells are deleted from the medullary collecting duct by two distinct mechanisms. However, the reason for the different modes of cell death is not known. As calbindin is reported to protect cells against apoptosis, we examined the expression of calbindin D28k in the developing rat kidney. Kidneys from 16-, 17-, 18- and 20-day-old fetuses and 1-, 3-, 5-, 7-, 14- and 21-day-old pups and adult Sprague Dawley rats were processed for immunohistochemistry using a monoclonal antibody against calbindin D28k. Intercalated cells were identified by immunostaining for H+-ATPase and by electron microscopy. Calbindin D28k immunoreactivity first appeared in subpopulations of cells in the connecting tubule and medullary collecting duct in the 17-day-old fetus. In the connecting tubule, calbindin D28k was expressed only in H+-ATPase negative connecting tubule cells, and there was no labeling of intercalated cells. In the medullary collecting duct, calbindin D28k immunostaining was observed in a few cells with apical H+-ATPase, characteristic of type A intercalated cells. The numbers of calbindin D28k-positive type A intercalated cells increased from day 18 of gestation. In contrast, there was little or no calbindin D28k immunoreactivity in the type B intercalated cells or principal cells. During the first two weeks after birth, calbindin D28k-positive type A intercalated cells were lost from the terminal part of the medullary collecting duct by simple extrusion. After two weeks, calbindin D28k immunostaining decreased in the type A intercalated cells throughout the medullary collecting duct. However, the immunoreactivity of calbindin D28k in the cortical collecting duct was increased in some of the type A intercalated cells and the adult pattern was observed in 21-day-old pups. Thus, we propose that the different expression of calbindin D28k in type A and type B intercalated cells may be responsible-at least partly-for the different modes of cell death demonstrated in these cells during kidney development.

장내분비 K-세포에서 인슐린-발현세포로의 분화

이에스더 ( Esder Lee ),유준모 ( Jun Mo Yu ),이민경 ( Min Kyung Lee ),류경렬 ( Gyeong Ryul Ryu ),고승현 ( Seung Hyun Ko ),안유배 ( Yu Bae Ahn ),문성대 ( Sung Dae Moon ),송기호 ( Ki Ho Song ) 대한당뇨병학회 2009 Diabetes and Metabolism Journal Vol.33 No.6

연구배경: 최근 제1형 당뇨병 치료법으로 췌도 이식의 새로운 발견에도 불구하고 췌장 공여자가 부족한 것이 여전히 중요한 장애물이다. 장상피에 존재하는 내분비세포(장내분비 세포)와 췌장 베타세포는 발생과정에서 유사한 분화경로를 공유한다. 특히, GIP를 분비하는 K-세포는 베타세포에서 관찰되는 중요한 단백질들이 많이 발현됨이 알려져있다. 따라서 저자들은 K-세포의 생물학적인 특성이 베타세포와 매우 유사하다는 점에 착안하여 이를 베타세포로 분화시키고자 본 연구를 시행하였다. 방법: 생쥐 췌장의 내분비 종양으로부터 기원한 복합 STC-1세포에서 K-세포를 순수 분리하였다. 또한, 안정적으로 Nkx6.1을 발현하는 K-세포 클론("Kn4-세포"로 명명)을 성공적으로 확보하였다. 혈청이 없는 조건에서 exendin-4를 처리함으로써 K-세포 또는 Kn4-세포가 시험관내에서 베타세포로 분화하도록 유도하였다. 인슐린 mRNA와 인슐린 단백질의 발현은 RT-PCR과 면역세포화학염색으로 조사하였다. 세포내 인슐린양도 측정하였다. 결과: K-세포에서 RT-PCR과 western blot 분석을 통하여 glucokinase와 GIP가 발현함을 확인하였다. RT-PCR의 결과에서 K-세포에서는 Pdx-1, NeuroD1/Beta2, MafA mRNA가 발현됨을 확인하였으나 Nkx6.1은 발현하지 않았다. Kn4-세포를 혈청이 없는조건에서 exendin-4을 처리한 결과, 인슐린 mRNA와 인슐린 또는 C-펩타이드 단백질이 뚜렷이 발현됨을 관찰하였다. 또한 세포내 인슐린양도 유의하게 증가하였다. 결론: K-세포는 특정한 조건에서 인슐린을 발현하는 세포로 분화가 되므로 장차 제1형 당뇨병의 세포치료법으로써 활용될 가능성이 있다. Background: Despite a recent breakthough in human islet transplantation for treating type 1 diabetes mellitus, the limited availability of donor pancreases remains a major obstacle. Endocrine cells within the gut epithelium (enteroendocrine cells) and pancreatic β cells share similar pathways of differentiation during embryonic development. In particular, K-cells that secrete glucose-dependent insulinotropic polypeptide (GIP) have been shown to express many of the key proteins found in β cells. Therefore, we hypothesize that K-cells can be transdifferentiated into β cells because both cells have remarkable similarities in their embryonic development and cellular phenotypes. Methods: K-cells were purified from heterogeneous STC-1 cells originating from an endocrine tumor of a mouse intestine. In addition, a K-cell subclone expressing stable Nkx6.1, called "Kn4-cells," was successfully obtained. In vitro differentiation of K-cells or Kn4-cells into β cells was completed after exendin-4 treatment and serum deprivation. The expressions of insulin mRNA and protein were examined by RT-PCR and immunocytochemistry. The interacellular insulin content was also measured. Results: K-cells were found to express glucokinase and GIP as assessed by RT-PCR and Western blot analysis. RT-PCR showed that K-cells also expressed Pdx-1, NeuroD1/Beta2, and MafA, but not Nkx6.1. After exendin-4 treatment and serum deprivation, insulin mRNA and insulin or C-peptide were clearly detected in Kn4-cells. The intracellular insulin content was also increased significantly in these cells. Conclusion: K-cells are an attractive potential source of insulin-producing cells for treatment of type 1 diabetes mellitus. However, more experiments are necessary to optimize a strategy for converting K-cells into β cells. (Korean Diabetes J 33:475-484, 2009)

Correlation analysis of cancer stem cell marker CD133 and human endogenous retrovirus (HERV)-K env in SKOV3 ovarian cancer cells

Kim Do-Ye,Kim Heungyeol,Ko Eun-Ji,Koh Suk Bong,Kim Hongbae,Lee Ji Young,Lee Chul Min,Eo Wan Kyu,Kim Ki Hyung,Cha Hee-Jae 한국유전학회 2024 Genes & Genomics Vol.46 No.4

Background Human endogenous retrovirus (HERV)-K is a type of retrovirus that is present in the human genome, and its expression is usually silenced in healthy tissues. The precise mechanism by which HERV-K env influences cancer stemness is not fully understood, but it has been suggested that HERV-K env may activate various signaling pathways that promote stemness traits in cancer cells. Objective To establish the connection between HERV-K env expression and cancer stemness in ovarian cancer cells, we carried out correlation analyses between HERV-K env and the cancer stem cell (CSC) marker known as the cluster of differentiation 133 (CD133) gene in SKOV3 ovarian cancer cells. Method To perform correlation analysis between HERV-K env and CSCs, ovarian cancer cells were cultured in a medium designed for cancer stem cell induction. The expression of HERV-K env and CD133 genes was verified using quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot analyses. Additionally, the expression of stemness-related markers, such as OCT-4 and Nanog, was also confirmed using RT-qPCR. Results In the stem cell induction medium, the number of tumorsphere-type SKOV3 cells increased, and the expression of CD133 and HERV-K env genes was up-regulated. Additionally, other stemness-related markers like OCT-4 and Nanog also exhibited increased expression when cultured in the cancer stem cell induction medium. However, when HERV-K env knockout (KO) SKOV3 cells were cultured in the same cancer stem cell induction medium, there was a significant decrease in the number of tumorsphere-type cells compared to mock SKOV3 cells subjected to the same conditions. Furthermore, the expression of CD133, Nanog, and OCT-4 did not show a significant increase in HERV-K env KO SKOV3 cells compared to mock SKOV3 cells cultured in the same cancer stem cell induction medium. Conclusion These findings indicate that the expression of HERV-K env increased in SKOV3 cells when cultured in cancer stem cell induction media, and cancer stem cell induction was inhibited by KO of HERV-K env in SKOV3 cells. These results suggest a strong association between HERV-K env and stemness in SKOV3 ovarian cancer cells. Background Human endogenous retrovirus (HERV)-K is a type of retrovirus that is present in the human genome, and its expression is usually silenced in healthy tissues. The precise mechanism by which HERV-K env influences cancer stemness is not fully understood, but it has been suggested that HERV-K env may activate various signaling pathways that promote stemness traits in cancer cells. Objective To establish the connection between HERV-K env expression and cancer stemness in ovarian cancer cells, we carried out correlation analyses between HERV-K env and the cancer stem cell (CSC) marker known as the cluster of differentiation 133 (CD133) gene in SKOV3 ovarian cancer cells. Method To perform correlation analysis between HERV-K env and CSCs, ovarian cancer cells were cultured in a medium designed for cancer stem cell induction. The expression of HERV-K env and CD133 genes was verified using quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot analyses. Additionally, the expression of stemness-related markers, such as OCT-4 and Nanog, was also confirmed using RT-qPCR. Results In the stem cell induction medium, the number of tumorsphere-type SKOV3 cells increased, and the expression of CD133 and HERV-K env genes was up-regulated. Additionally, other stemness-related markers like OCT-4 and Nanog also exhibited increased expression when cultured in the cancer stem cell induction medium. However, when HERV-K env knockout (KO) SKOV3 cells were cultured in the same cancer stem cell induction medium, there was a significant decrease in the number of tumorsphere-type cells compared to mock SKOV3 cells subjected to the same conditions. Furthermore, the expression of CD133, Nanog, and OCT-4 did not show a significant increase in HERV-K env KO SKOV3 cells compared to mock SKOV3 cells cultured in the same cancer stem cell induction medium. Conclusion These findings indicate that the expression of HERV-K env increased in SKOV3 cells when cultured in cancer stem cell induction media, and cancer stem cell induction was inhibited by KO of HERV-K env in SKOV3 cells. These results suggest a strong association between HERV-K env and stemness in SKOV3 ovarian cancer cells.

HLA-restricted and Antigen-specific CD8+ T Cell Responses by K562 Cells Expressing HLA-A*0201

Yun, Sun-Ok,Sohn, Hyun-Jung,Yoon, Sung-Hee,Choi, Hee-Baeg,Kim, Tai-Gyu The Korean Association of Immunobiologists 2006 Immune Network Vol.6 No.4

Background: Identification of antigen-specific T cells has yielded valuable information on pathologic process and the disease state. Assays for quantification of inflammatory cytokines or lytic-granule molecules have been generally used to evaluate antigen specific T cell response, however their applicability have been hampered due to the limited source of autologous antigen-presenting target cells (APC). Methods: K562, a leukemic cell line deficient of human leukocyte antigen (HLA), was transfected with a gene encoding HLA-A*02 (K562/ A*02) and its function as stimulator cells in inducing activation of HLA-matched T cells was evaluated by IFN-${\gamma}$ enzyme linked immunospot (ELISPOT) assay. Results: The stable transfectant K562/ A*02 pulsed with HLA- A*02 restricted peptide could specifically induce IFN-${\gamma}$ secretion by CD8+ T cells compared to no detectable secretion by CD4+ T cells. However, CD56+ NK cells secreted IFN-${\gamma}$ in both K562/ A*02 with peptide and without peptide. The number of IFN-${\gamma}$ secreted CD8+ T cells was increased according to the ratio of T cells to K562 and peptide concentration. Formalin-fixed K562/ A*02 showed similar antigen presenting function to live K562/ A*02. Moreover, K562/ A*02 could present antigenicpeptide to not only A*0201 restricted CD8+ T cells but also CD8+ T cells from A*0206 donor. Conclusion: These results suggest that K562/ A*02 could be generally used as target having specificity and negligible background for measuring CD8+ T cell responses and selective use of K562 with responsder matched HLA molecules on its surface as APC may circumvent the limitation of providing HLA-matched autologous target cells.

K562 백혈병 세포주에서 방사선에 의해 유도되는 Apoptosis에 미치는 PTK Inhibitors의 영향

이형식(Hyung Sik Lee),문창우(Chang Woo Moon),허원주(Won Joo Hur),정수진(Su Jin J eong),정민호(Min Ho Jeong),이정현(Jeong Hyeon Lee),임영진(Young Jin Lim),박헌주(Heon Joo Pa rk) 대한방사선종양학회 2000 Radiation Oncology Journal Vol.18 No.1

목 적 : 방사선에 의해 유도되는 apoptos is에 내성을 가진 세포로 알려진 K562 세포를 대상으로, PTK inhibitors인 herbimycin A와 genist ein을 이용한 방사선에 의한 apoptos is의 내성 기전을 연구하고자 하였다. 대상 및 방법 : 6 MV 체외 X-선 방사선 치료기를 이용하여 200∼300 cGy/min의 선량률로 10 Gy의 X-선을 세포에 균일하게 조사하였다. Apoptosis의 관찰은 agarose gel elect rophores is를 이용하여 DNA frgment at ion의 지표인 ladder를 관찰하였고, TUNEL 염색을 이용하여 정량 분석을 시행하였다. Western blot 방법으로 apoptos is 관련 유전 단백인 bcl-2, bcl-XL 및 bax들의 발현을 관찰하였다. 방사선 조사 및 약물 처치 후의 세포 주기 분석은 flow cytometry로 분석하였다. 결 과 :Agarose gel elect rophores is 실험에서 방사선을 조사하지 않은 K562 세포와 방사선을 10 Gy 조사한 세포를 48시간에 걸쳐 12시간 간격으로 관찰하였을 때 DNA fragment at ion를 관찰할 수 없었다. 이러한 현상은 genistein을 투여한 세포들에서도 동일한 현상을 관찰할 수 있었지만, 방사선 조사 후 herbimycin A를 투여한 세 포들에서는 48시간째 확연한 DNA fragmentation을 관찰할 수 있었다. 이를 TUNEL assay에서 정량적으로 확인하였다. 방사선만 조사한 세포들과 방사선과 genistein 투여 후 48시간째 관찰한 세포들에서는 10%미만의 apoptosis 양성 세포의 빈도를 관찰할 수 있었지만, 방사선 조사 후 herbimycin A를 투여한 세포들에서는 30∼35%빈도로 apoptosis 양성 세포들이 관찰되었다. Western blot analysis에서 bcl-2의 경우 방사선을 조사하지 않았던 대조군에 비하여 전체적인 발현은 증가되었지만 방사선 및 약제간의 발현의 차이는 없었다. 그 외 bcl-XL과 bax는 대조군에 비해 방사선 및 약제간의 발현의 차이를 관찰할 수 없었다. K562 세포에 방사선을 10 Gy 조사하였을 때 나타나는 세포 주기의 변화는 시간이 경과함에 따라 전형적인 G2/Mblock의 소견을 보였다. 이러한 소견은 genistein을 투여했을 경우에는 특별한 변화를 보이지 않지만, herbimycin A를 투여했을 경우에는 12시간째부터 G2/Mblock이 소실되면서 세포가 세포 주기를 재 순환하는 양상을 보였고, 48시간째 관찰한 소견에서는 G2/Mblock이 거의 소실된 양상을 띠었다. 이러한 소견을 토대로 apoptosis 유도와의 상호 연관성을 유추할 수 있었다. 결 론 : herbimycin A는 방사선에 의해 유도되는 apoptosis가 억제된 K562 세포에서 apoptosis를 유도할 수 있었다. 이러한 유도 기전에 apoptos is 관련 유전 단백들인 bcl-2, bcl-XL 및 bax와 관련된 영향은 관찰되지 않았다. 세포 주기의 분석에서 G2/Mblock의 해소와 apoptos is 유도와의 연관성을 유추할 때, 세포 주기 관련 인자들에 대한 연구가 방사선에 의한 apoptosis의 내성의 극복 및 방사선에 의한 세포의 감수성 조절 약제로서의 역할에 이바지할 것으로 생각한다. Purpose :The effect of PTK inhibitors (herbimycin A and genistein) on the induction of radiation-induced apoptosis in Ph- positive K562 leukemia cell line was investigated. Materials and Methods :K562 cells in exponential growth phase were irradiated with a linear accelerator at room temperature. For 6 MV X- ray irradiation and drug treatment, cultures were initiated at 2x106cells/mL. The cells were irradiated with 10 Gy. Stock solutions of herbimycin A and genistein were prepared in dimethyl sulphoxide (DMSO). After incubation at 37℃ for 0∼48 h, the extent of apoptosis was determined using agarose gel electrophoresis and TUNEL assay. The progression of cells through the cell cycle after irradiation and drug treatment was also determined with flow cytometry. Western blot analysis was used to monitor bcl-2, bcl-XL and bax protein levels. Results :Treatment with 10 Gy X- irradiation did not result in the induction of apoptosis. The HMA alone (500 nM) also failed to induce apoptosis. By contrast, incubation of K562 cells with HMA after irradiation resulted in a substantial induction of nuclear condensation and fragmentation by agarose gel electrophoresis and TUNEL assay. Genistein failed to enhance the ability of X- irradiation to induce DNA fragmentation. Enhancement of apoptosis by HMA was not attributable to downregulation of the bcl- 2 or bcl-XL anti- apoptotic proteins. When the cells were irradiated and maintained with HMA, the percentage of cells in G2/M phase decreased to 30∼40% at 48 h. On the other hand, cells exposed to 10 Gy Xirradiation alone or maintained with genistein did not show marked cell cycle redistribution. Conclusion :We have shown that nanomolar concentrations of the PTK inhibitor HMA synergize with X- irradiation in inducing the apoptosis in Ph (+) K562 leukemia cell line. While, genistein, a PTK inhibitor which is not selective for p210bc r/a bl failed to enhance the radiation induced apoptosis in K562 cells. It is unlikely that the ability of HMA to enhance apoptosis in K562 cells is attributable to bcl- 2 family. It is plausible that the relationship between cell cycle delays and cell death is essential for drug development based on molecular targeting designed to modify radiation- induced apoptosis.

제1형 당뇨병 쥐 모델에서 유전공학적 제조 K-세포 이식을 통한 당뇨병의 치료

심주연 ( Ju Yeon Sim ),김주희 ( Ju Hee Kim ),안유배 ( Yu Bae Ahn ),송기호 ( Ki Ho Song ),한제호 ( Je Ho Han ),차봉연 ( Bong Yun Cha ),이숙경 ( Sook Kyung Lee ),문성대 ( Sung Dae Moon ) 대한당뇨병학회 2009 Diabetes and Metabolism Journal Vol.33 No.6

연구배경: 인슐린 유전자 치료법의 이상적인 표적세포로 K-세포가 알려져 있다. 이전 연구에서 본 연구자들은 EBV-유래 에피솜 벡터를 이용하여 K-세포에서 포도당농도 의존적인 인슐린 분비가 이루어지는 것을 실험실 환경에서 확인한 바 있다. 본 연구에서는 스트렙토조토신(STZ)으로 유발된 제1형 당뇨병 쥐의 신장 피막에 유전공학적으로 제조된 K-세포를 이식하여 당뇨병이 치료되는지를 관찰해 보았다. 방법: 인슐린이 분비되도록 제조된 K-세포를 STZ으로 유도된 BALB/c Nude 마우스의 신장 피막에 이식한 후 혈당과 몸무게를 측정하였다. 마우스는 정상군과 STZ으로 유도된 당뇨병군 그리고 당뇨병군의 일부는 K-세포를 이식한 치료군 등으로 분류하여 실험을 하였다. 이식 4주 후 모든 마우스에서 6시간 동안 금식한 후 2 g/kg의 포도당을 복강내로 주사하여 당내성검사를 하였다. 당내성검사가 끝나자마자 모든 마우스의 신장과 췌장을 적출하여 인슐린, 글루카곤, C-peptide 등으로 면역조직화학염색 및 면역형광염색을 한 후 그 결과를 분석하였다. 결과: STZ을 복강에 주사한 마우스는 주사 3일 후 혈당이 300±50 mg/dL로 상승하였다. 정상군과 K-세포를 이식한 당뇨병군에서는 몸무게가 점차 증가하였으나, STZ만을 주사한 당뇨병군에서는 실험기간 동안 낮게 유지되었다. 그리고 정상군에서는 정상범위의 혈당분포를 유지하였으나, STZ만을 주사한 당뇨병군에서는 지속적으로 혈당이 높게 유지되었다. 그리고 K-세포를 이식한 당뇨병군에서는 STZ만을 주사한 군에서보다 혈당이 서서히 떨어졌으며 이식 2주 후부터는 급격히 감소하였다. 당부하검사 결과 정상군과 K-세포를 이식한 당뇨병군에서는 서로 유사한 내당력을 보였으나 STZ만을 주사한 군에서는 지속적으로 포도당 불내성을 보였다. 조직면역검사 결과 정상군의 췌장에서는 인슐린과 글루카곤, C-peptide 모두에 염색이 되었지만 STZ만을 주사한 마우스의 췌장에서는 췌도파괴로 인슐린은 물론 글루카곤, C-peptide 모두에 염색이 되지 않았다. 그러나, K-세포를 이식한 당뇨병군의 신장에서는 면역조직화학염색에서 인슐린과 C-peptide 모두에서 염색이 되었으며 면역형 광염색에서도 인슐린이 발현하는 것을 관찰할 수 있었다. 결론: 유전공학적으로 제조된 K-세포가 마우스 신장 피막에 착상되어 혈당농도 의존적으로 인슐린을 분비하여 당뇨병이 치료되는 것을 생체 내에서도 확인할 수 있었으며, EBV-유래 에피솜벡터를 이용한 유전공학적 조제 K-세포는 제1형 당뇨병의 치료를 위한 대체베타세포로 사용될 수 있을 것으로 생각된다. Background: K-cells function as targets for insulin gene therapy. In a previous study, we constructed EBV-based plasmids expressing rat preproinsulin controlled by glucose-dependent insulinotropic polypeptide promoters. In the present study, we attempted to correct hyperglycemia in vivo using genetically engineered K-cells in a mouse model of type 1 diabetes. Methods: K-cells expressing insulin were transplanted under the kidney capsules of STZ-induced diabetic mice. The blood glucose levels and body weights of the experimental animals were measured daily. After four weeks, the mice were injected intra-peritoneally with 2 g/kg glucose following a 6 hr fast. Blood glucose levels were measured immediately following glucose injections. All animals were sacrificed at the end of the glucose tolerance study, and pancreas and graft-bearing kidney tissue samples were stained with antibodies against insulin, glucagon, and C-peptide. Results: The body weights of K-cell-transplanted diabetic mice increased after transplantation, whereas those of untreated diabetic control mice continued to decline. The blood glucose levels of K-cell-transplanted diabetic mice decreased gradually during the two weeks following transplantation. After intra-peritoneal injection of glucose into K-cell-transplanted diabetic mice, blood glucose levels increased at 30 minutes, and were restored to the normal range between 60 and 90 minutes, while untreated control diabetic mice continued to experience hyperglycemia. Kidney capsules containing transplanted K-cells were removed, and sections were stained with anti-insulin antibodies. We detected insulin-positive cells in the kidney capsules of K-cell-transplanted diabetic mice, but not in untreated control mice. Conclusion: We detected glucose-dependent insulin secretion in genetically engineered K-cells in a mouse model of type 1 diabetes. Our results suggest that genetically modified insulin producing K-cells may act as surrogate β-cells to effectively treat type 1 diabetes. (Korean Diabetes J 33:466-474, 2009)

Types and density of calbindin D28k-immunoreactive ganglion cells in mouse retina

Gu, Y.N.,Lee, E.S.,Jeon, C.J. Academic Press 2016 Experimental eye research Vol.145 No.-

Single-cell injection after immunocytochemistry is a reliable technique for classifying neurons by their morphological structure and their expression of a particular protein. The aim of the present study was to classify the morphological types of calbindin D28k-immunoreactive retinal ganglion cells in the mouse using single-cell injection after immunocytochemistry, to estimate the density of calbindin D28k-immunoreactive retinal ganglion cells in the mouse retina. Calbindin D28k is an important calcium-binding protein that is widely expressed in the central nervous system. Calbindin D28k-immunoreactive retinal ganglion cells were identified by immunocytochemistry and then iontophoretically injected with the lipophilic dye, DiI. Subsequently, the injected cells were imaged by confocal microscopy to classify calbindin D28k-immunoreactive retinal ganglion cells based on their dendritic ramification depth within the inner plexiform layer, field size, and morphology. The cells were heterogeneous in morphology: monostratified or bistratified, with small to large dendritic field size and sparse to dense dendritic arbors. At least 10 different morphological types (CB1-CB10) of calbindin D28k-immunoreactive retinal ganglion cells were found in the mouse retina. The density of each cell type was quite variable (1.98-23.76%). The density of calbindin D28k-immunoreactive cells in the ganglion cell layer of the mouse retina was 562 cells/mm<SUP>2</SUP>, 8.18% of calbindin D28k-immunoreactive cells were axon-less displaced amacrine cells, 91.82% were retinal ganglion cells, and approximately 18.17% of mouse retinal ganglion cells expressed calbindin D28k. The selective expression of calbindin D28k in cells with different morphologies may provide important data for further physiological studies of the mouse retina.

Combination of Potassium Pentagamavunon-0 and Doxorubicin Induces Apoptosis and Cell Cycle Arrest and Inhibits Metastasis in Breast Cancer Cells

Putri, Herwandhani,Jenie, Riris Istighfari,Handayani, Sri,Kastian, Ria Fajarwati,Meiyanto, Edy Asian Pacific Journal of Cancer Prevention 2016 Asian Pacific journal of cancer prevention Vol.17 No.5

A salt compound of a curcumin analogue, potassium pentagamavunon-0 (K PGV-0) has been synthesized to improve solubility of pentagamavunon-0 which has been proven to have anti-proliferative effects on several cancer cells. The purpose of this study was to investigate cytotoxic activity and metastasis inhibition by K PGV-0 alone and in combination with achemotherapeutic agent, doxorubicin (dox), in breast cancer cells. Based on MTT assay analysis, K PGV-0 showed cytotoxic activity in T47D and 4T1 cell lines with $IC_{50}$ values of $94.9{\mu}M$ and $49.0{\pm}0.2{\mu}M$, respectively. In general, K PGV-0+dox demonstrated synergistic effects and decreased cell viability up to 84.7% in T47D cells and 62.6% in 4T1 cells. Cell cycle modulation and apoptosis induction were examined by flow cytometry. K PGV-0 and K PGV-0+dox caused cell accumulation in G2/M phase and apoptosis induction. Regarding cancer metastasis, while K PGV-0 alone did not show any inhibition of 4T1 cell migration, K PGV-0+dox exerted inhibition. K PGV-0 and its combination with dox inhibited the activity of MMP-9 which has a pivotal role in extracellular matrix degradation. These results show that a combination of K PGV-0 and doxorubicin inhibits cancer cell growth through cell cycling, apoptosis induction, and inhibition of cell migration and MMP-9 activity. Therefore, K PGV-0 may have potential for development as a co-chemotherapeutic agent.

Melatonin-induced calbindin-D9k expression reduces hydrogen peroxide-mediated cell death in rat pituitary GH3 cells

Yoo, Yeong-Min,Jeung, Eui-Bae Blackwell Publishing Ltd 2010 Journal of pineal research Vol.48 No.2

<P>Abstract: </P><P>In this study, we investigated whether calbindin-D9k (CaBP-9k) expression was regulated by melatonin during hydrogen peroxide (H<SUB>2</SUB>O<SUB>2</SUB>)-induced cell death in rat pituitary GH3 cells. CaBP-9k expression was increased by melatonin in a dose- and time-dependent manner, indicating that CaBP-9k expression is regulated by melatonin. Cell survival was increased approximately 27–30% where H<SUB>2</SUB>O<SUB>2</SUB>-treated cells (0.25 or 0.5 m<SMALL>M</SMALL>) were also incubated with 1 m<SMALL>M</SMALL> melatonin, when compared with H<SUB>2</SUB>O<SUB>2</SUB> alone or H<SUB>2</SUB>O<SUB>2</SUB> plus 0.5 m<SMALL>M</SMALL> melatonin. This result was consistent with 4,6-diamidino-2-phenylindole staining. CaBP-9k expression was also augmented by co-treatment with H<SUB>2</SUB>O<SUB>2</SUB> and 1 m<SMALL>M</SMALL> melatonin, suggesting a functional relationship between increased cell death and melatonin-induced CaBP-9k expression during H<SUB>2</SUB>O<SUB>2</SUB>-mediated apoptosis. Bcl-2-associated protein expression increased following treatment with H<SUB>2</SUB>O<SUB>2</SUB> alone, whereas Bcl-2 expression was elevated following treatment with melatonin alone, or H<SUB>2</SUB>O<SUB>2</SUB> plus melatonin. The expression of p53 was depressed by treatment with melatonin alone, or co-treatment with H<SUB>2</SUB>O<SUB>2</SUB> plus melatonin. These results correlated with CaBP-9k expression levels and activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway. Knockdown of CaBP-9k expression using a small inhibitory RNA resulted in an elevation of H<SUB>2</SUB>O<SUB>2</SUB>-induced cell death, whereas cell survival was increased in cells that overexpressed CaBP-9k, providing additional evidence that the induction of CaBP-9k expression may be associated with survival signaling during H<SUB>2</SUB>O<SUB>2</SUB>-mediated oxidative cell death. CaBP-9k appears to interact with p53, suggesting a possible role for this interaction in cell proliferation and cell cycle progression.</P>