KR101749218B1 - Human adipocyte conditioned media extract derived from adipose stem cells having hair growth-promoting effects and uses thereof - Google Patents
Human adipocyte conditioned media extract derived from adipose stem cells having hair growth-promoting effects and uses thereof Download PDFInfo
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- KR101749218B1 KR101749218B1 KR1020100038540A KR20100038540A KR101749218B1 KR 101749218 B1 KR101749218 B1 KR 101749218B1 KR 1020100038540 A KR1020100038540 A KR 1020100038540A KR 20100038540 A KR20100038540 A KR 20100038540A KR 101749218 B1 KR101749218 B1 KR 101749218B1
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- derived stem
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- stem cells
- human adipose
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Abstract
본 발명은 인간 지방유래 줄기세포를 무혈청 배지에서 배양한 후, 여과를 하여 얻은 조건 배지를 유효성분으로 함유하는 탈모예방 또는 양모유도용 약학 조성물, 상기 약학 조성물을 탈모부위 또는 무모 부위에 국소적으로 도포 또는 피내주사하여 양모를 유도하는 방법, 상기 약학 조성물을 유효성분으로 포함하는 탈모 치료제 및 무모증 치료제에 관한 것이다.The present invention relates to a pharmaceutical composition for hair loss prevention or wool induction, which comprises culturing human adipose-derived stem cells in a serum-free medium and then containing the conditioned medium obtained by filtration as an active ingredient, A method of inducing wool by applying or intradermally injecting the composition, a hair loss therapeutic agent containing the above-mentioned pharmaceutical composition as an active ingredient, and an anti-hair remedy.
Description
본 발명은 양모효과를 가지는 인간 지방유래 줄기세포의 배양액 및 이의 용도에 관한 것으로, 더욱 상세하게는 인간 지방유래 줄기세포를 무혈청 배지에서 배양한 후, 여과를 하여 얻은 조건 배지를 유효성분으로 함유하는 탈모예방 또는 양모유도용 약학 조성물, 상기 약학 조성물을 탈모부위 또는 무모 부위에 국소적으로 도포 또는 피내주사하여 양모를 유도하는 방법, 상기 약학 조성물을 유효성분으로 포함하는 탈모 치료제 및 무모증 치료제에 관한 것이다.The present invention relates to a culture solution of human adipose-derived stem cells having a wool effect and, more particularly, to a method for culturing human adipose-derived stem cells in a serum-free medium, A method of inducing wool by topically applying or intradermally injecting the pharmaceutical composition to a hair loss site or a hairless area, a hair loss treatment agent containing the pharmaceutical composition as an active ingredient, and an anti-hair treatment agent will be.
최근 높아진 미용에 대한 관심 때문에 탈모증의 치료에 대한 대중의 관심 또한 높아지고 있다. 탈모증(alopecia)이란 비정상적으로 털이 많이 빠지는 증상을 말하며, 대게는 머리털이 많이 빠지는 증상을 말한다. 모발은 발생, 성장, 퇴화 및 휴지기의 주기를 가지는데 휴지기에 있는 모발이 하루에 보통 50 내지 100개 정도가 정상적으로 빠지는 것으로 알려졌다. Due to recent interest in beauty, public interest in the treatment of alopecia is also growing. Alopecia (alopecia) refers to symptoms of abnormal hair loss, usually a lot of hair loss symptoms refers to. Hair has a cycle of development, growth, degeneration and dormancy, and it is known that the hair in the dormant period normally suffers from about 50 to 100 times a day.
탈모증의 원인에는 여러 가지가 있는데, 산모의 출산 후 후유증, 스트레스, 갑상선기능항진증(hyperthyroidism) 또는 갑상선기능저하증(hypothyroidism), 과도한 다이어트, 철 결핍(iron deficiency), 항암치료 후유증, 남성호르몬성 탈모(androgenetic alopecia) 및 두피백선과 같은 피부병 등이 알려져 있다. There are many causes of alopecia, which include maternal aftereffects, stress, hyperthyroidism or hypothyroidism, excessive diet, iron deficiency, sequelae of chemotherapy, male hormonal hair loss androgenetic alopecia) and skin diseases such as scalp ringworm are known.
탈모의 치료법으로 현재 가장 많이 사용하는 방법은 프로페시아(propecia)의 경구투여 및 미녹시딜(minoxidil)의 국소적 도포와 같은 약물요법이며, 그외에 수술적 방법으로 자기 자신의 모발을 이용하여 이식하는 자가모발이식이 널리 알려져 있다. 프로페시아는 합성 안티안드로젠(anti-androgen)으로서, 테스토스테론(testosterone)을 디하이드로테스토스테론(dihydrotestosterone, DHT)으로 전환시키는 효소인 5-알파 환원효소(5-alpha reductase)를 억제한다. 상기 약물은 원래 양성 전립선비대증(benign prostatic hyperplasia) 및 전립선암(prostate cancer)의 치료에 사용되어 왔으며, 그 후에 남성호르몬성 탈모에도 효과가 있는 것으로 알려져 1997년 남성형 탈모치료제로 미국 식품의약국의 승인을 받았다. 하지만, 프로페시아를 지속적으로 복용할 경우 남성에서 발기부전(erectile dysfunction) 및 성기능 장애와 같은 부작용이 있을 수 있으며, 임산부 및 가임기 여성의 피부에 흡수될 경우 태아의 웅성생식기 기형을 초래할 수도 있는 것으로 알려졌다. 미녹시딜은 혈관확장제 및 탈모증의 치료에 쓰이며, 부작용으로는 두피 가려움증이 있다. 상기 약물요법에 의한 치료는 투여할 당시에는 효과가 나타나지만 치료가 중단되면 탈모가 다시 진행되는 것으로 밝혀졌다. 자가모발이식 또한 반영구적이라는 장점이 있지만, 비교적 많은 비용이 소모된다는 단점이 있다. Currently, the most commonly used method of treating hair loss is oral administration of propecia and topical application of minoxidil. In addition, surgical methods such as hair transplantation using their own hair Transplantation is widely known. Propecia is a synthetic anti-androgen that inhibits the enzyme 5-alpha reductase, which converts testosterone to dihydrotestosterone (DHT). The drug has been originally used for the treatment of benign prostatic hyperplasia and prostate cancer and has been known to be effective against male hormonal hair loss afterwards. In 1997, the drug was approved by the US Food and Drug Administration . However, continued use of Propecia may have adverse effects such as erectile dysfunction and sexual dysfunction in men, and may result in fetal male reproductive anomalies when absorbed into the skin of pregnant women and women of childbearing age. Minoxidil is used for the treatment of vasodilators and alopecia, with side effects such as scalp itching. Treatment with the drug regimen is effective at the time of administration, but it turns out that hair loss resumes once treatment is stopped. Although hair transplantation is also semi-permanent, it has a disadvantage that it is relatively expensive.
한국공개특허 제2008-0097593호에는 인간 지방조직 유래 성체줄기세포 및 모낭세포를 함유하는 세포치료제가 개시되어 있으며, 한국등록특허 제10-0771171호에는 모낭 줄기세포를 유효성분으로 하는 대머리 치료용 조성물이 개시되어 있다. Korean Patent Publication No. 2008-0097593 discloses a cell therapy agent containing human adipose tissue-derived adult stem cells and hair follicle cells. In Korean Patent No. 10-0771171, a composition for treating baldness comprising follicular stem cells as an active ingredient .
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명자들은 인간 지방유래 줄기세포를 무혈청 배지에서 배양한 후, 여과를 통해 얻은 조건 배지가 표피 및 진피의 구성세포인 불사화 케라틴 형성세포 및 인간 진피유두세포를 효과적으로 증식시키는 것을 발견하고 본 발명을 완성하게 되었다.The present inventors have found that stem cells derived from human adipose are cultured in a serum-free medium, and then conditioned medium obtained by filtration is composed of a non-oxidative keratin-forming cell, which is a constituent cell of epidermis and dermis, Human papillary papilloma cells, thereby completing the present invention.
상기 과제를 해결하기 위해, 본 발명은 인간 지방유래 줄기세포를 무혈청 배지에서 배양한 후, 여과를 하여 얻은 조건 배지를 유효성분으로 함유하는 탈모예방 또는 양모유도용 약학 조성물을 제공한다.In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing hair loss or inducing wool, which comprises culturing human adipose-derived stem cells in a serum-free medium and then filtering the hair-derived stem cells as an active ingredient.
또한, 본 발명은 상기 약학 조성물을 탈모부위 또는 무모 부위에 국소적으로 도포 또는 피내주사하는 것을 특징으로 하는 양모를 유도하는 방법을 제공한다. In addition, the present invention provides a method of inducing wool characterized in that the pharmaceutical composition is locally applied or injected intradermally at a hair loss site or a hairless area.
또한, 본 발명은 상기 약학 조성물을 유효성분으로 포함하는 탈모 치료제를 제공한다. The present invention also provides a therapeutic agent for depilation comprising the above pharmaceutical composition as an active ingredient.
또한 본 발명은 상기 약학 조성물을 유효성분으로 포함하는 무모증 치료제를 제공한다. The present invention also provides an anti-aspiring agent comprising the pharmaceutical composition as an active ingredient.
본 발명의 인간 지방유래 줄기세포의 조건 배지는 피부조직의 재생 및 양모를 유도하는 활성을 가지므로 탈모증의 치료와 예방에 효과적으로 사용될 수 있을 것으로 기대된다.The conditional medium of human adipose-derived stem cells of the present invention is expected to be effectively used for the treatment and prevention of alopecia due to the regeneration of skin tissue and the activity of inducing wool.
도 1은 인간 진피유두세포(human dermal papilla cells)를 인간 지방유래 줄기세포-조건 배지 및 지방세포-조건 배지로 처리한 후, MTT 분석법에 의한 세포증식의 측정결과를 나타낸다. 분리된 인간 지방유래 줄기세포(adipose tissue-derived stem cells, ADSCs)는 9, 12 및 15일 동안 α-MEM에서 유지되었다. 인간 지방유래 줄기세포에서 지방세포로의 분화는 지방세포분화배지(인슐린, 덱사메사손, 이소부틸-메틸-크산틴(isobutyl-methyl-xanthine) 및 인도메사신)에 의해 9, 12 및 15일 동안 유도되었다. 인간 지방유래 줄기세포-조건 배지 및 분화된 지방세포-조건 배지는 무혈청 α-MEM에서 48시간 배양 후 각각 수집되었다. 인간 진피유두세포는 인간 지방유래 줄기세포-조건 배지 및 지방세포-조건 배지에서 배양되었고, 24시간 및 48시간 후 MTT 분석법에 의해 세포의 증식이 측정되었다. 48시간 동안 인간 지방유래 줄기세포-조건 배지를 처리한 군에서 인간 진피유두세포의 증식이 유의적으로 증가하였고, 반면에 지방세포-조건 배지의 처리는 증식을 억제하였다. 스튜던트 T 검정이 통계적 분석에 이용되었고, 모든 결과들은 적어도 3번 이상의 독립적인 실험을 통한 평균으로 나타내었다. 또한 각각의 독립적인 실험들을 위해 인간 지방유래 줄기세포의 다른 공급원(sources)이 사용되었다. *: p < 0.05 에서 음성대조군에 대한 유의성 검정, d: 각 배지에서의 처리기간.
도 2는 인간 지방유래 줄기세포-조건 배지 처리 후, 인간 진피유두세포에서 신호전달 단백질들의 발현 변화를 나타낸다. 인간 지방유래 줄기세포-조건 배지 처리 후, 인산화된 Akt의 발현은 미세하게 증가하였고, 인산화된 Erk의 발현은 명확하게 증가하였다.
도 3은 인간 지방유래 줄기세포-조건 배지 처리 후, 인간 진피유두세포의 세포주기 및 세포주기 관련 단백질을 분석한 결과를 나타낸다. 인간 지방유래 줄기세포-조건 배지는 세포주기의 조절을 통해 인간 진피유두세포의 증식을 증가시켰다. 48시간의 인간 지방유래 줄기세포-조건 배지의 처리는 대조군에 비해 S기의 증가 및 G1기 정체(arrest)의 감소를 야기시켰다(상단). 인간 지방유래 줄기세포-조건 배지 처리 24시간 후, Cyclin D 및 CDK2의 발현은 명확하게 상향조절(up-regulation) 되었다(하단). CNT: 대조군, ADSC-CM: 인간 진피유두세포에 처리한 인간 지방유래 줄기세포-조건 배지.
도 4는 불사화 케라틴 형성 세포에 인간 지방유래 줄기세포-조건 배지를 처리한 후, 불사화 케라틴 형성 세포의 증식을 MTT 분석법으로 측정한 결과를 나타낸다. 불사화 케라틴 형성세포를 24시간 동안 스타빙(starving)한 후, 인간 지방유래 줄기세포-조건 배지에 48시간 동안 배양한다. 이어서 MTT 분석법에 의해 세포의 증식을 측정하였다. 인간 지방유래 줄기세포-조건 배지의 처리는 10 내지 30%의 농도에서 유의적으로 불사화 케라틴 형성 세포의 증식을 증가시켰다. 모든 결과들은 적어도 3번 이상의 독립적인 실험을 통한 평균으로 나타내었다. 또한 각각의 독립적인 실험들을 위해 인간 지방유래 줄기세포의 다른 공급원(sources)이 사용되었다. 스튜던트 T 검정이 통계적 분석에 이용되었다. *: p < 0.05 에서 음성대조군에 대한 유의성 검정.
도 5는 남성(n=5, 평균나이=34.8세)의 후두부에서 수집한 ex vivo 성장기 모발(anagen hair organ)에서 모발성장에 대한 인간 지방유래 줄기세포-조건 배지의 영향을 나타낸다. 12.5%의 인간 지방유래 줄기세포-조건 배지가 Williams E 배지에 첨가되어 졌다. 음성 대조군으로서 1/2 부피의 α-MEM이 Williams E 배지에 혼합되어졌으며 양성 대조군으로서 미녹시딜(minoxidil) 1 mM이 Williams E 배지에 첨가되어졌다. 12.5%의 인간 지방유래 줄기세포-조건 배지를 처리한 군에서 유의적인 모간 신장(hair shaft elongation)이 관찰되었다. 결과 수치(values)는 각 성장 조건당 15개 이상의 모낭(hair follicle)의 누적 성장(cumulative growth)을 6일 동안 측정하여 평균±표준오차로 나타내었다. 스튜던트 T 검정이 통계적 분석에 이용되었다. 5개의 인간 지방유래 줄기세포-조건 배지가 5명의 남성 두피 샘플에 임으로 각각 적용되었다. *: p < 0.05 에서 음성대조군에 대한 유의성 검정.
도 6은 C3H/HeN 누드마우스에서 인간 지방유래 줄기세포에 의한 모발 성장 자극을 나타낸다. 7주령 C3H/HeN 누드마우스를 이용하여 모발성장의 유도가 측정되었으며 모낭들은 휴지기로 동질화(synchronization)되었다. 인간 지방유래 줄기세포(5 x 105 cells) 및 대조군인 PBS(phosphate buffered saline)가 9일 동안 3일 간격으로 마우스의 등쪽 피부에 피내주사(intradermal injection) 되었다. 모발의 성장은 첫 번째 주사 후 12주 동안 측정되었다. 그 결과 PBS를 처리한 대조군에 비해 인간 지방유래 줄기세포를 처리한 군에서 모발성장이 증가한 것을 관찰하였다. 국소적인 적용을 한 군에서는 피부의 흡수율을 높이기 위해 0.2 mm 메소롤러(mesoroller; Moohan, Korea)를 처리 전에 10회 사용하였다. 모발성장 자극효과는 피부의 다크닝(darkening)에 의해 측정되었다. 12주 후, 대조군에 비해 인간 지방유래 줄기세포-조건 배지를 국소적으로 적용한 군에서 모발성장이 증가되었다.
도 7은 대조군 및 인간 지방유래 줄기세포-조건 배지를 처리한 C3H/HeN 누드마우스의 등쪽 피부에 대한 H&E 염색 결과를 나타낸다. 조직학적 평가는 인간 지방유래 줄기세포-조건 배지의 첫 번째 국소적인 적용 2 및 4주 후에 수행되었다. 그 결과 인간 지방유래 줄기세포-조건 배지 처리 4주 후에 모낭의 수가 증가된 것을 관찰하였다. FIG. 1 shows the results of measurement of cell proliferation by MTT assay after treatment of human dermal papilla cells with human adipose-derived stem cell-conditioned medium and adipocyte-conditioned medium. Separated adipose tissue-derived stem cells (ADSCs) were maintained in α-MEM for 9, 12 and 15 days. Differentiation into adipocytes from human adipose-derived stem cells was induced by adipocyte differentiation medium (insulin, dexamethasone, isobutyl-methyl-xanthine and indomethacin) for 9, 12 and 15 days . Human adipose-derived stem cells-conditioned medium and differentiated adipocyte-conditioned medium were collected after 48 hour incubation in serum-free alpha-MEM, respectively. Human dermal papilla cells were cultured in human adipose-derived stem cell-conditioned medium and adipocyte-conditioned medium, and cell proliferation was measured by MTT assay at 24 and 48 hours. The proliferation of human dermal papilla cells was significantly increased in the group treated with human adipose - derived stem cell - conditioned media for 48 hours, while the treatment of adipocyte - conditioned media inhibited proliferation. Student's T test was used for statistical analysis and all results were expressed as an average over at least three independent experiments. Different sources of human adipose derived stem cells were also used for each independent experiment. *: Significance test for negative control at p < 0.05, d: duration of treatment in each medium.
Figure 2 shows the expression changes of signal transduction proteins in human dermal papilla cells after human adipose-derived stem cell-conditioned medium treatment. After human adipose-derived stem cell-conditioned medium treatment, the expression of phosphorylated Akt was increased slightly and the expression of phosphorylated Erk was clearly increased.
FIG. 3 shows the results of analysis of cell cycle and cell cycle-related proteins of human dermal papilla cells after treatment with human adipose-derived stem cell-conditioned medium. Human adipose-derived stem cell-conditioned media increased the proliferation of human dermal papilla cells through regulation of the cell cycle. Treatment of human adipose-derived stem cell-conditioned medium for 48 hours caused an increase in S phase and a decrease in G1 arrest (upper) compared to the control group. After 24 h of human adipose-derived stem cell-conditioned medium, the expression of Cyclin D and CDK2 was clearly up-regulated (bottom). CNT: control, ADSC-CM: human adipose-derived stem cell-conditioned medium treated with human dermal papilla cells.
FIG. 4 shows the results of measuring the proliferation of the immortalized keratin-forming cells by MTT assay after treating the human-derived stem cell-conditioned medium with the non-keratinized cells. Immortalized keratin-forming cells are starved for 24 hours and then cultured in human adipose-derived stem cell-conditioned medium for 48 hours. Cell proliferation was then measured by MTT assay. Treatment of human adipose-derived stem cell-conditioned media significantly increased the proliferation of non-keratinized cells at concentrations of 10-30%. All results were expressed as averages over at least 3 independent experiments. Different sources of human adipose derived stem cells were also used for each independent experiment. Student's T test was used for statistical analysis. *: Significance test for negative control at p < 0.05.
FIG. 5 is a graph showing the relationship between ex (n = 5, average age = 34.8 years) collected at the occiput of a male induced adipocyte stem cell-conditioned media for hair growth in vivo growing anagen hair organs. 12.5% human adipose-derived stem cell-conditioned medium was added to Williams E medium. As a negative control, 1/2 volume of? -MEM was mixed in Williams E medium and 1 mM of minoxidil as a positive control was added to Williams E medium. Significant hair shaft elongation was observed in the group treated with 12.5% human adipose-derived stem cell-conditioned medium. The results are expressed as mean ± standard error (cumulative growth) of more than 15 hair follicles per growth condition for 6 days. Student's T test was used for statistical analysis. Five human adipose-derived stem cell-conditioned media were applied to five male scalp samples, respectively. *: Significance test for negative control at p < 0.05.
Figure 6 shows hair growth stimulation by human adipose-derived stem cells in C3H / HeN nude mice. The induction of hair growth was measured using a 7 week old C3H / HeN nude mouse and the hair follicles were synchronized to the resting period. Human adipose-derived stem cells (5 x 10 5 cells) and control group PBS (phosphate buffered saline) were injected intradermally into the dorsal skin of mice at intervals of 3 days for 9 days. Hair growth was measured for 12 weeks after the first injection. As a result, hair growth was observed in the group treated with human adipose tissue stem cells compared to the control group treated with PBS. In the topical application group, a 0.2 mm meso roller (Moohan, Korea) was used 10 times before treatment to increase the uptake of the skin. The hair growth stimulating effect was measured by darkening of the skin. After 12 weeks, hair growth was increased in the topically applied group of human adipose derived stem cell-conditioned medium compared to the control group.
Figure 7 shows H & E staining results for dorsal skin of C3H / HeN nude mice treated with control and human adipose-derived stem cell-conditioned medium. Histological evaluation was performed 2 and 4 weeks after the first topical application of human adipose-derived stem cell-conditioned medium. As a result, we observed an increase in the number of hair follicles after 4 weeks of human fat-derived stem cell-conditioned medium treatment.
본 발명의 목적을 달성하기 위하여, 본 발명은 인간 지방유래 줄기세포를 무혈청 배지에서 배양한 후, 여과를 하여 얻은 조건 배지(conditioned medium)를 유효성분으로 함유하는 탈모예방 또는 양모유도용 약학 조성물을 제공한다.In order to accomplish the object of the present invention, the present invention relates to a pharmaceutical composition for preventing hair loss or for inducing wool, which comprises culturing human adipose-derived stem cells in a serum-free medium and then filtering the conditioned medium obtained as an active ingredient .
본 발명의 약학 조성물에서, 상기 조건 배지는 바람직하게는 인간 지방유래 줄기세포를 무혈청 α-MEM(modified Eagle medium)에서 40 내지 60시간 동안 배양한 후, 원심분리한 다음 0.2 내지 0.25μm 필터로 여과를 하여 얻을 수 있으며, 더욱 바람직하게는 인간 지방유래 줄기세포를 무혈청 α-MEM(modified Eagle medium)에서 48시간 동안 배양한 후, 원심분리한 다음 0.22μm 필터로 여과를 하여 얻을 수 있다.In the pharmaceutical composition of the present invention, the conditioned medium is preferably cultured human serum-derived stem cells in serum-free modified Eagle medium (α-MEM) for 40 to 60 hours, centrifuged and then filtered with 0.2 to 0.25 μm filter Filtration. More preferably, the human adipose-derived stem cells are cultured in serum-free modified Eagle medium for 48 hours, centrifuged, and then filtered through a 0.22-μm filter.
본 발명의 약학 조성물에서, 상기 조건 배지는 인간 진피유두세포(human dermal papilla cell)와 인간 케라티노세포(human keratinocytes)의 증식을 증가시킴으로써 두피 및 모발성장을 촉진할 수 있다.In the pharmaceutical composition of the present invention, the conditioned medium can promote scalp and hair growth by increasing the proliferation of human dermal papilla cells and human keratinocytes.
상기 줄기세포란 여러 종류의 조직 세포로 분화할 수 있는 능력을 가진 미분화 세포를 말하며, 배반포(blastocyst)의 내세포괴(inner cell mass)로부터 분리되는 배아줄기세포(embryonic stem cell) 및 성체 조직으로부터 분리되는 성체줄기세포(adult stem cell)로 크게 분류할 수 있다. 성체줄기세포는 인간을 포함한 포유동물, 바람직하게는 인간의 성체 조직으로부터 유래된 다분화능(mutipotency)을 갖는 미분화세포를 말하며, 예를 들어, 골수, 혈액, 뇌, 피부, 지방, 제대혈 등의 다양한 성체세포로부터 유래될 수 있다. The stem cells are undifferentiated cells capable of differentiating into various types of tissue cells, and they are separated from embryonic stem cells and adult tissues isolated from inner cell mass of blastocyst. (Adult stem cells) can be roughly classified into. Adult stem cells refer to undifferentiated cells having multipotency derived from mammals including humans, preferably human adult tissues. For example, adult stem cells include various cells such as bone marrow, blood, brain, skin, Can be derived from adult cells.
상기 인간 지방유래 줄기세포는 인간의 지방조직으로부터 분리된 일종의 성체줄기세포이다. 지방조직의 획득은 통상적으로 시행되는 지방흡입(liposuction) 과정에서 부수적으로 얻을 수 있어 충분한 양의 줄기세포를 용이하게 얻고 배양할 수 있다는 장점이 있으며, 또한 골수 채취에 비해 안전성이 우수하다. The human adipose-derived stem cells are a kind of adult stem cells isolated from human adipose tissue. The acquisition of adipose tissue can be obtained incidentally during the liposuction process, which is usually performed, and thus it is possible to easily obtain and cultivate a sufficient amount of stem cells, and also has superior safety compared to bone marrow harvesting.
상기 인간 지방유래 줄기세포는 줄기세포 배양용 배지, 예를 들어 혈청 배지를 이용하여 통상의 방법으로 배양될 수 있다. 바람직하게는 인간 지방유래 줄기세포를 10% 소태아혈청을 함유하는 α-modified Eagle medium에서 배양한 후, 최종적으로 무혈청 α-modified Eagle medium에서 배양함으로써, 획득되는 배양액 중의 단백질 함량을 높일 수 있다. 상기 무혈청 배지 단계는 지방조직으로부터 분리한 줄기세포를 특정의 극한 환경에 노출시킴으로서 분화를 최소화함과 더불어 줄기세포가 분비하는 단백질을 배양액으로부터 최대한 많이 회수할 수 있게 한다.The human adipose-derived stem cells can be cultured by a conventional method using a medium for stem cell culture, for example, a serum medium. Preferably, human adipose-derived stem cells are cultured in α-modified Eagle medium containing 10% fetal bovine serum and finally cultured in serum-free α-modified Eagle medium to increase protein content in the obtained culture medium . The serum-free medium step minimizes differentiation by exposing the stem cells isolated from adipose tissue to a specific extreme environment, and enables the recovery of the proteins secreted by the stem cells from the culture medium as much as possible.
본 발명의 조건 배지를 포함하는 약학조성물은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있다.The pharmaceutical compositions comprising the conditioned media of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the preparation of pharmaceutical compositions.
본 발명의 조건 배지의 약학적 투여 형태는 이들의 약학적 허용 가능한 염의 형태로도 사용될 수 있고, 또한 단독으로 또는 타 약학적 활성 화합물과 결합뿐만 아니라 적당한 집합으로 사용될 수 있다.The pharmaceutical dosage forms of the conditioned media of the present invention may also be used in the form of their pharmaceutically acceptable salts and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable set.
본 발명에 따른 조건 배지를 포함하는 약학조성물은, 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 조건 배지를 포함하는 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유 등을 포함한 다양한 화합물 혹은 혼합물을 들 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 추출물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트 (calcium carbonate), 수크로스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈 (tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.The pharmaceutical composition comprising the conditioned medium according to the present invention may be formulated into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions Can be used. Examples of carriers, excipients and diluents that can be contained in the composition containing the conditioned medium include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Various compounds or mixtures including silicates, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.
본 발명의 조건 배지의 바람직한 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. 그러나, 바람직한 효과를 위해서, 본 발명의 조건 배지는 1일 0.0001 내지 100 mg/kg으로, 바람직하게는 0.001 내지 100 mg/kg으로 투여하는 것이 좋다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위을 한정하는 것은 아니다.The preferable dosage of the condition medium of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the administration route and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the condition medium of the present invention is preferably administered at 0.0001 to 100 mg / kg per day, preferably 0.001 to 100 mg / kg per day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.
본 발명의 조건 배지는 쥐, 생쥐, 가축, 인간 등의 포유동물에 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구, 직장 또는 정맥, 근육, 피하, 국소적 도포 또는 피내주사에 의해 투여될 수 있다.Conditioned media of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in a variety of routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, muscular, subcutaneous, topical application or intradermal injection.
또한, 본 발명은 상기 약학 조성물을 탈모부위 또는 무모 부위에 국소적으로 도포 또는 피내주사하는 것을 특징으로 하는 양모를 유도하는 방법을 제공한다. 상기 투여 형태는 바람직하게는 국소적 도포 또는 피내주사일 수 있으나, 이에 제한되지 않는다.In addition, the present invention provides a method of inducing wool characterized in that the pharmaceutical composition is locally applied or injected intradermally at a hair loss site or a hairless area. The dosage form may be, but is not limited to, topical application or intradermal injection.
또한, 본 발명은 상기 약학 조성물을 유효성분으로 포함하는 탈모 치료제 및 무모증 치료제를 제공한다.In addition, the present invention provides a depilatory remedy comprising the above-mentioned pharmaceutical composition as an active ingredient, and an anti-hair remedy.
본 발명의 탈모 치료제 및 무모증 치료제는 남성호르몬성 탈모증 및 폐경 또는 난소제거 수술 후 발생할 수 있는 여성형 탈모증에 대한 치료를 위하여 두피에 적용할 수 있을 뿐만 아니라 발모를 필요로 하는 신체 부위라면 어디나 적용할 수 있다.The hair loss therapeutic agent and the treatment for alopecia of the present invention can be applied not only to the scalp but also to any part of the body where hair growth is necessary for the treatment of female haploid alopecia that may occur after male hormone alopecia and postmenopausal or ovariectomy surgery have.
본 발명의 탈모 치료제 및 무모증 치료제에 포함되는 약제학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. The pharmaceutically acceptable carrier to be included in the depilatory remedy and the asymmetry treatment agent of the present invention is one which is commonly used in the present invention and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, Gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, But is not limited thereto.
본 발명의 탈모 치료제 및 무모증 치료제는 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다. The depilatory remedy and the asymmetry remedy of the present invention can be formulated into a unit dose form and / or an asymmetrical form by using a pharmaceutically acceptable carrier and / or an excipient according to a method which can be easily carried out by those having ordinary skill in the art to which the present invention belongs. Or may be prepared by entrapping in a multi-dose container, and may additionally include a dispersant or stabilizer.
본 발명의 탈모 치료제 및 무모증 치료제는 단독의 요법으로 이용될 수 있으나, 다른 통상적인 발모 유도 약물요법 또는 수술요법 등과 함께 이용될 수도 있으며, 이러한 병행요법을 실시하였을 경우 극대화된 효과를 나타낼 수 있다.
The hair loss remedy and the asymmetry remedy of the present invention may be used as a single therapy but may be used together with other conventional hair growth induction drug therapy or surgery therapy and the maximized effect can be exhibited when such concurrent therapy is performed.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.
실시예Example 1: 인간 지방유래 줄기세포( 1: human adipose-derived stem cells ( ADSCsADSCs )의 배양 및 조건 배지() And conditioned medium ( ConditionedConditioned media)의 수집 media collection
인간 지방유래 줄기세포(ADSC)는 이전에 기재된 바와 같이 배양되어졌다(Lee et al., 2004, Cell Physiol biochem 14, 311-324). 인간 지방유래 줄기세포는 평균나이 43.8세의 여성 5명으로부터 지방흡입(liposuction)에 의해 획득된 피하지방조직(subcutaneous adipose tissue)으로부터 수집하였다. 인간 지방유래 줄기세포는 상기 피하지방조직에 제1형 콜라겐 분해효소(collagenase type I, Sigma-Aldrich, St.Louis, MO, USA) 0.075%를 37℃에서 45분간 처리한 후, 70μm 메쉬 필터(mesh filter)로 여과하여 분리되었고, 이어서 상기 여과물질을 α-modified Eagle medium (α-MEM; Invitrogen, Carlsbad, CA, USA)에 혼합하고 원심분리하였다. 획득된 펠렛부 즉, 인간 지방유래 줄기세포 분획(fraction)을 세척한 후, 1200×g 에서 5분 동안 원심분리하였고, 이어서 10%의 소태아혈청(fetal bovine serum, FBS)을 함유하는 α-MEM에 상기 분획물을 재부유하여 37℃ 5% CO2에서 배양하였다. 인간 지방유래 줄기세포에서 지방세포로의 분화(adipogenic differentiation)는 인슐린(insulin), 덱사메사손(dexamethasone), 이소부틸-메틸-크산틴(isobutyl-methyl-xanthine), 및 인도메사신(indomethacin)에 의해 9 내지 15일 동안 유도되었다. Human adipose derived stem cells (ADSCs) were cultured as previously described (Lee et al., 2004, Cell Physiol Biochem 14, 311-324). Human adipose derived stem cells were collected from subcutaneous adipose tissue obtained by liposuction from five women of average age 43.8 years. Human fat-derived stem cells were treated with 0.075% collagenase type I collagenase type I (Sigma-Aldrich, St. Louis, Mo., USA) for 45 minutes at 37 ° C, mesh filter, and then the filtrate was mixed with α-modified Eagle medium (α-MEM; Invitrogen, Carlsbad, CA, USA) and centrifuged. The obtained pellet portion, that is, a human adipose-derived stem cell fraction, was centrifuged at 1200 xg for 5 minutes, and then an α-lactamase containing 10% fetal bovine serum (FBS) The fractions were resuspended in MEM and incubated at 37 ° C in 5% CO 2 . Adipogenic differentiation from human adipose-derived stem cells into adipocyte differentiation leads to insulin, dexamethasone, isobutyl-methyl-xanthine, and indomethacin. RTI ID = 0.0 > 9-15 < / RTI >
상기 배양세포로부터 조건 배지(conditioned media, CM)를 수확하기 위하여, 인간 지방유래 줄기세포(5 × 105 cells)와 성숙한 지방세포(adipocytes)를 무혈청(serum-free) α-MEM에서 배양하였다. 인간 지방유래 줄기세포-조건 배지(ADSC-CM)와 지방세포-조건 배지(adipocyte-CM)를 배양 48시간 후에 수집하였고, 수집한 조건 배지들은 원심분리한 다음 0.22 μm의 시린지 필터(syringe filter)로 여과하였다.
In order to harvest the condition medium (conditioned media, CM) from the cultured cells, human adipose derived stem cells (5 × 10 5 cells) and mature fat cells (adipocytes) were cultured in serum-free (serum-free) α-MEM . Human adipose-derived stem cell-conditioned medium (ADSC-CM) and adipocyte-CM medium were harvested 48 hours after culture. The collected conditioned mediums were centrifuged and resuspended in 0.22 μm syringe filter. Lt; / RTI >
실시예Example 2: 인간 2: human 진피유두세포Dermal papilla cells (( humanhuman dermaldermal papillapapilla cellscells ) 및 ) And 불사화Immortalization 케라틴 형성 세포의 증식( Proliferation of keratinocytes proliferationproliferation ) 및 세포주기 분석) And cell cycle analysis
모발의 주기변화는 표피와 진피 성분들의 빠른 리모델링(remodeling)을 포함하므로, 배양된 인간 진피유두세포(human dermal papilla cells, hDPCs) 및 불사화 케라틴 형성세포(immortalized keratinocyte; HaCaT cell)의 증식(proliferation)을 조사하였다. 인간 진피유두세포와 불사화 케라틴 형성 세포는 10% 소태아혈청을 함유한 Dulbecco's modified Eagle's medium (DMEM; Invitrogen-Gibco-BRL)에서 배양되었다. 상기 세포의 증식은 인간 지방유래 줄기세포-조건 배지를 처리한 후, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide를 이용한 MTT 분석법(assay)에 의해 측정되었다. 또한 각각의 독립적인 실험들을 위해 인간 지방유래 줄기세포의 다른 공급원(sources)이 사용되었다. 인간 지방유래 줄기세포-조건 배지를 25, 50 및 75%의 농도로 처리한 군에서는 인간 진피유두세포의 증식이 최대 130%까지 증가된 반면, 지방세포-조건 배지의 첨가는 증식을 억제하였다(도 1).Since the periodic change of hair involves rapid remodeling of epidermal and dermal components, the proliferation of cultured human dermal papilla cells (hDPCs) and immortalized keratinocyte (HaCaT cells) ) Were investigated. Human dermal papilla cells and non-keratinized cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen-Gibco-BRL) containing 10% fetal bovine serum. The cell proliferation was assessed by MTT assay using 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide after treating human adipose stem cell-conditioned medium Lt; / RTI > Different sources of human adipose derived stem cells were also used for each independent experiment. Proliferation of human dermal papilla cells was increased up to 130% in the groups treated with human fat-derived stem cell-conditioned medium at 25, 50 and 75%, while addition of adipocyte-conditioned medium inhibited proliferation 1).
기초가 되는 메카니즘을 명확하게 하기 위해, 세포의 증식과 관련있는 신호전달 단백질들이 웨스턴 블롯(Western blot)에 의해 분석되었다. 인산화된Akt(pAkt; Ser473, Cell Signaling Tech., Inc., MA), Akt(Cell Signaling Tech., Inc., MA) 및 인산화된 Erk(pErk; Thr202/Tyr204, Cell Signaling Tech., Inc., MA)에 대한 1차 항체가 사용되었다. 웨스턴 블롯의 결과에서, 인산화된 Akt의 발현 수준(level)은 총(total) Akt에 비해 인간 지방유래 줄기세포-조건 배지 처리 24 및 48시간 후에 증가하였다. 인산화된 Erk 발현 또한 총 Erk의 발현이 변하지 않은 동안, 인간 지방유래 줄기세포-조건 배지 처리에 따라 확연하게 증가하였다(도 2). To clarify the underlying mechanism, signaling proteins associated with cell proliferation were analyzed by Western blot. (Cell Signaling Tech., Inc., MA), Akt (Cell Signaling Tech., Inc., MA) and phosphorylated Erk (pErk; Thr202 / Tyr204, Cell Signaling Tech. MA) was used. In the Western blot results, the levels of phosphorylated Akt expression were increased 24 and 48 hours after the human adipose-derived stem cell-conditioned medium compared to total Akt. Phosphorylated Erk expression was also significantly increased following treatment with human adipose-derived stem cell-conditioned media, while the expression of total Erk was unchanged (Figure 2).
다음으로 인간 지방유래 줄기세포-조건 배지를 처리한 인간 진피유두세포의 세포주기 분석이 유세포 분석기(FACScan flow cytometer, Beckton-Dickinson, CA)를 사용하여 수행되었다. G1기 정체(G1 arrest)를 시키기 위해, 인간 진피유두세포(3.2 × 105 cells/dish)은 스타빙(starving) 되어졌고, 이어서 배지는 무혈청 배지 또는 50%의 인간 지방유래 줄기세포-조건 배지로 교체되어 48시간 동안 배양되었다. FACS 세포주기분석 결과에서, 인간 지방유래 줄기세포-조건 배지의 처리는 인간 진피유두세포의 세포성장기(G1)는 감소시킨 반면, DNA 합성기(S) 및 유사분열기(G2-M)는 증가시켰다(도 3, 상단). 이어서 수행한 웨스턴 블롯에서, p21, p27 및 CDK4의 발현은 변화되지 않았지만(데이타 미제시), 세포주기와 관계되는 중요한 분자인 cyclin D1(Serotec, UK) 및 CDK2(Santa Cruz biotechnology)는 인간 지방유래 줄기세포-조건 배지 처리 24시간 후에 명확히 상향조절(up-regulation) 되었다(도 3, 하단). 상기 결과들은 인간 지방유래 줄기세포-조건 배지가 세포주기의 조절을 통하여 인간 진피유두세포의 증식을 증가시킨다는 것을 나타낸다. 게다가 인간 지방유래 줄기세포-조건 배지는 Erk 및 Akt 신호전달 경로를 활성화함으로써 진피유두세포의 생존과 증식을 촉진하는 것으로 나타난다. 진피 유두의 크기는 모발의 성장주기(hair growth cycle)와 밀접하게 관계되어 있으며, 진피 유두의 세포수는 모발 성장기(anagen phase)에서 증가한다고 보고되어 있다(Elliott et al., 1999, J Invest Dermatol 113, 873-877). 또한, 인간 지방유래 줄기세포-조건 배지는 불사화 케라틴 형성 세포의 증식을 10 내지 30%의 농도에서 유의적으로 촉진한다(도 4).
Next, cell cycle analysis of human dermal papilla cells treated with human adipose-derived stem cell-conditioned medium was performed using a flow cytometer (FACScan flow cytometer, Beckton-Dickinson, Calif.). To induce G1 arrest, human dermal papilla cells (3.2 x 10 5 cells / dish) were starved and the medium was then grown in serum-free medium or 50% human adipose-derived stem cell-conditioned And then cultured for 48 hours. In the FACS cell cycle analysis, treatment of human adipose-derived stem cell-conditioned medium increased the growth of the human dermal papilla cells (G1) while increasing the DNA synthesizer (S) and mitotic factor (G2-M) 3, top). Cyclin D1 (Serotec, UK) and CDK2 (Santa Cruz biotechnology), important molecules involved in the cell cycle, did not change the expression of p21, p27 and CDK4 in subsequent western blots Was clearly up-regulated 24 hours after stem cell-conditioned medium treatment (Fig. 3, bottom). These results indicate that human adipose-derived stem cell-conditioned medium increases the proliferation of human dermal papilla cells through regulation of the cell cycle. In addition, human adipose-derived stem cell-conditioned medium appears to promote survival and proliferation of dermal papillary cells by activating the Erk and Akt signaling pathways. The size of the dermal papilla is closely related to the hair growth cycle and the number of dermal papillary cells is reported to increase in the anagen phase (Elliott et al., 1999, J Invest Dermatol 113, 873-877). In addition, the human adipose-derived stem cell-conditioned medium significantly promotes the proliferation of the immortalized keratin-forming cells at a concentration of 10-30% (FIG. 4).
실시예Example 3: 모간 신장( 3: Moraine kidney ( hairhair shaftshaft elongationelongation )에 대한 인간 지방유래 줄기세포-조건 배지) Human adipose-derived stem cells-conditioned medium 의of 효과 분석 Effect analysis
인간 지방유래 줄기세포-조건 배지가 현저한 유사분열(mitogenic) 효과를 나타내므로, 모간 신장(hair shaft elongation)에 대한 인간 지방유래 줄기세포-조건 배지의 효과가 조사되었다. 5명의 남성(평균나이 34.8세, 한 명당 70 내지 80개의 모낭)으로부터 수집된 총 370개의 성장기 모낭(anagen hair follicles)이 하이드로코티손(hydrocortisone) 10 ng/mL, 인슐린 10 g/mL, L-글루타민 2 mM 및 페니실린 100 U/mL을 함유하는 Williams E 배지(Gibco BRL, Gaithersburg, MD, USA)에서 37℃, 5% CO2 조건하에 배양되었다. Williams E 배지에 인간 지방유래 줄기세포-조건 배지를 12.5%, 25%, 37.5% 또는 50% 수준으로 첨가한 후, 6일 동안 배양하였다. 그 결과, 12.5% 처리군에서 모낭의 길이가 대조군에 비해 40%까지 유의적으로 신장되었으며, 이는 미녹시딜(minoxidil) 1 mM로 처리한 양성 대조군보다 더 높았다(도 5).
The effect of human adipose-derived stem cell-conditioned medium on hair shaft elongation was investigated as the human adipose-derived stem cell-conditioned medium exhibits a marked mitogenic effect. A total of 370 anagen hair follicles collected from 5 men (mean age 34.8 years old, 70 to 80 hair follicles per person) received 10 ng / mL hydrocortisone, 10 g / mL insulin, L- glutamine (Gibco BRL, Gaithersburg, Md., USA) containing 5% CO 2 , 2 mM and penicillin 100 U / Lt; / RTI > Human adipogenic stem cell-conditioned medium was added to Williams E medium at the level of 12.5%, 25%, 37.5% or 50%, and cultured for 6 days. As a result, the hair follicle length was significantly increased by 40% in the 12.5% treated group compared with the control group treated with minoxidil 1 mM (Fig. 5).
실시예Example 4: 4: 생체내(in vivo)에서In vivo 인간 지방유래 줄기세포-조건 배지의 양모효과 분석 Analysis of the wool effect of human adipose-derived stem cells-conditioned media
7주령 수컷 C3H/HeN 누드마우스(n=48, Orient Bio, Korea)의 등쪽 피부의 털을 면도하고 모낭을 휴지기(telogen stage)로 동질화(synchronization)하였다. 이어서 인간 지방유래 줄기세포(5 x 105 cells/50 mL PBS) 또는 PBS(phosphate buffered saline)를 9일 동안 3일 간격으로 등쪽 피부에 피내주사(intradermal injection)하였다. 동시에 인간 지방유래 줄기세포-조건 배지 1 mL 또는 대조군 배지를 다른 C3H 마우스의 등에 국소적으로 도포하였다. 그 결과, 인간 지방유래 줄기세포를 피내주사한 C3H/HeN 누드마우스의 모낭은 휴지기에서 성장기로의 전환이 대조군에 비해 빨리 유도되었으며(도 6, 상단), 조건 배지를 국소도포한 군에서는 모발의 성장이 촉진됨을 발견하였다(도 6, 하단). 또한 인간 지방유래 줄기세포-조건 배지를 처리한 마우스의 등쪽 피부를 조직학적으로 살펴보았을 때 모낭의 수가 증가함을 발견하였다(도 7). 상기 결과들은 국소적으로 주사된 인간 지방유래 줄기세포 및 인간 지방유래 줄기세포-조건 배지가 생체내(in vivo)에서 모발의 성장을 촉진할 수 있다는 것을 가리킨다. 인간 지방유래 줄기세포에서 분비되는 인자가 모발성장에 다소간 치료 잠재성을 가지는 것으로 생각된다. Seven week old male C3H / HeN nude mice (n = 48, Orient Bio, Korea) shaved the dorsal skin and synchronized the hair follicle with the telogen stage. Subsequently, intradermal injection of human adipose-derived stem cells (5 x 10 5 cells / 50 mL PBS) or PBS (phosphate buffered saline) into the dorsal skin was performed at intervals of 3 days for 9 days. At the same time, 1 mL of human fat-derived stem cell-conditioned medium or control medium was topically applied to the back of another C3H mouse. As a result, the hair follicle of the C3H / HeN nude mouse injected intracutaneously with human adipose-derived stem cells was faster in the transition from dormant to growth phase than the control (Fig. 6, top) (Fig. 6, bottom). It was also found that when the dorsal skin of mice treated with human adipose-derived stem cell-conditioned medium was histologically examined, the number of hair follicles was increased (Fig. 7). These results indicate that locally injected human adipose-derived stem cells and human adipose-derived stem cell-conditioned medium can promote hair growth in vivo. It is thought that factors secreted from human adipose derived stem cells have somewhat therapeutic potential for hair growth.
본 발명의 결과들은 인간 지방유래 줄기세포가 인간 진피유두세포의 증식을 증가시킴으로서 모발성장을 촉진하며, 표피세포에서도 세포주기의 조절 및 모발주기의 성장기(anagen phase)를 활성화함으로써 가능하게 한다는 것을 나타낸다. 따라서 인간 지방유래 줄기세포의 합리적인 조작은 모발성장촉진을 위한 하나의 좋은 수단으로 생각된다. The results of the present invention show that human adipose-derived stem cells promote hair growth by increasing proliferation of human dermal papilla cells, and also enable epidermal cells to regulate the cell cycle and activate the anagen phase of the hair cycle . Therefore, rational manipulation of human adipose-derived stem cells is considered to be a good means for promoting hair growth.
Claims (6)
상기 조건 배지는 인간 지방유래 줄기세포를 무혈청 α-MEM(modified Eagle medium)에서 40 내지 60시간 동안 배양한 후, 원심분리한 다음 0.2 내지 0.25μm 필터로 여과를 하여 얻은 것을 특징으로 하는 탈모예방 또는 양모유도용 약학 조성물.Human adipose-derived stem cells were cultured in a serum-free medium, and then 12.5% by weight of a conditioned medium obtained by filtration was contained as an active ingredient,
The conditional medium was obtained by culturing human adipose-derived stem cells in serum-free modified medium (α-MEM) for 40 to 60 hours, followed by centrifugation followed by filtration through a 0.2 to 0.25 μm filter. ≪ / RTI >
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