AMANO, TAKEHIKO

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Title CYTOTOXIC ACTION OF THE VACCINE AND THEKOKTIGEN TO THE CHICKEN FIBROBLAST IN VITROAuthor(s)AMANO, TAKEHIKOCitation日本外科宝函 (1962), 31(4): 635-645Issue Date1962-07-01URL http://hdl.handle.net/2433/205457RightType Departmental Bulletin PaperTextversionpublisherKyoto University635 CYTOTOXIC ACTION OF THE VACCINE AND THE KOKTIGEN TO THE CHICKEN FIBROBLAST IN VITRO by T AKEHIKO AMANO From the 2nd Surgical Division Kyoto University Medical School (Director : Prof邸 orDr. YASUMASA Aov AGI) Received for Puhlication Apr. 16, 1962 INTRODUCTION Many investigations have been made in the £eld of vaccinization and its application as a prevention against infectious diseases. With the improvement of chemotherapeutics, the bacterial infectious dis回sessuch as typhoid fever and dysentery have markedly de-creased in the last ten years. But recently, the bacterial infections by the chemothera-petics resistant bacteria are increasing, and this phenomenon is going to be a serious problem in the public health. It is nec白saryto prevent the infectious diseases both by drugs and the natural and aquired resistance in the body. This paper d田lswith the toxic action of heat-killed bacterial vaccine at 60 ° C and 100。Cto the chicken五broblastin vitro. Materials and Methods The first experiment Preparations of heat-killed bacterial solutions : Salmonella. typhosa H. 901 strain of the Microbiological laboratory was used. The bacterial colonies which had been incubated on the nutrient agar during the period of eighteen or twenty hours were collected with the standard platinum string (int he inner volume of its ring equivalent to五vemg. of bacteria in wet weight). This bacterial mass was dissolved in Hanks solution (pH 7.4），五lteredthrough gauze into uniform and difuse bacterial suspension. These StJSpensions were heated in a water bath at a tern-perature of 100。Cand 60° C each. In the preparation of bacterial五ltrate,these heated bacterial suspensions were centri-fuged at 3000 r.p.m. for 30 minutes and its supernatant were used. From eggs incubated for ten days, the chick embryos were removed aseptically and their hearts were cut into small pieces 1×Imm¥ arranged three in a row on each cover slide (22×40mm) , then inserted into each test tube. These test tubes were placed in the thermostat at 37° C for 30 minutes and finally poured into these test tubes with .4 cc of culture media of Hanks solution 80 % and bovine serum 20 % . After incubation of 48 hours at 37°C, 4 cc of the experimental culture media containing 20% bovine serum were exchanged. On the 1st, 3rd, and 5th day after mediae change, the inserted slides were removed from the test tubes and observed by pha~e contrast microscope. In each different experimental media, five test tubes were prepared. The experimental culture media were as follows . 636 日本外科宝盛l第31巻第4号1 A). Four cc of Hanks solution 80 %, bovine serum 20 % (Hanks 80 Bs 20) containing heat-killed Salmonella typhosa 1 mg per ml. (l00°C for 30 minutes) 1 A’） . Four cc of Hanks 80 Bs 20 containing heat-killed Salmonella typhosa lmg per ml. (60° C for 30 minutes) 1 B). Four cc of Hanks 80 Bs 20 containing Salmonella typhosa O.lmg per ml. (100。cfor 30 minutes) 1 B’） . Four cc of Hanks 80 Bs 20 containing Salmonella 0.1 mg per ml. (60°C for 30 minutes) 1 C). Four cc of Hanks 80 Bs 20 containing Salmonella typhosa 0.01 mg (100。cfor 30 minutes) 1 c’） . Four cc of Hanks 80 Bs 20 containing Salmonella typhosa 0.01 mg (60°C for 30 minutes) As control, four cc of Hanks 80 Bs 20 was used. The culture mediae of the bacterial filtrate of each dilution were as follows : 2 A) Four cc of Hanks solution 80% of the supernatant of the bacterial suspension containing 1 25 mg of heat-killed Salmonella typhosa at 100。Cfor 30 minutes and bovine serum 20% (1 mg of Salmonella typhosa per 1 ml of culture media) 2 A’） Four αof Hanks solution 80% of the supernatant of the bacterial suspension containing 1.25 mg of heat-killed Salmonella typhosa at 60°C for 30 minutes and bovine serum 20%. 2 B) Four cc of Hanks 80 of the supernatant of the bacterial suspension containing 0.125 mg of heat-killed Salmonella typhosa at 100°C for 30 minutes and Bs 20. 2 B’） Four cc of Hanks 80 of the supernatant of the becterial suspension contain-ing 0.125 mg of heat-killed Salmonella typhosa at 60°C for 30 minutes and Bs 20. 2 C) Four cc of Hanks 80 of the supernatant of the bacterial suspension contain-ing 0.0125 mg of heat-killed Salmonella typhosa at 100。cfor 30 minutes and Bs 20. 2 c’） Four cc of Hanks 80 of the supernatant of the bacterial suspension contain-ing 0.0125 mg of heat-killed Salmonella typhosa at 60°C for 30 minutes and Bs 20. THE SECOND EXPERI恥tlE恥tlTTo estimate the experiment quantitatively, the dispersed and monolayer culture by Y oungner method were used. Ten-day-chick embryos were cut with scissors and this material was trypsinized in Hanks solution containing 0.25 % trypsin of Difco, and the dispersed cels were collected and separated by centrifugation at 1000 r.p.m. for 10 minutes, counted in a cel count with 0.1 % citric crystal violet and finally adjusted 5×10弓 per2 ml of Hanks 80 Bs 20 containing different amount of heat-killed Salmonella typhosa vaccine with supernatant of the bacterial suspension in t白ttube by stationary culture method. On the 1st, 3rd and 5th day after incubation the cultured cels were measured by trypsinization and citric crystal violet method. The culture media which were used in the second experiment was the same as those of the first experiment. 3 A) Two cc of Hanks 80% Bs 20 % containing heat-killed Salmonella typhosa 1 mg. per ml. (100°C for 30 minutes) 637 3A’） Two cc of Hanks 80 Bs 20 containing heat-kiled Salmonella typhosa 1 mg per ml. (60°C for 30 minutes) 3 B) Two cc of Hanks 80 Bs 20 containing Salmonella typhosa (100°C for 30 minutes) 3 B’） Two cc of Hanks 80 Bs 20 containing (60°C for 30 minutes) 3 C) Two cc of Hanks 80 Bs 20 containing Salmonella typhosa for 30 minutes) 3 C') Two cc of Hanks 80 Bs 20 containing Salmonella typhosa for 30 minutes) 4 A) Two cc of Hanks 80 of the supernatant of the bacterial suspension contain-ing 1.25 mg of heat-killed Salmonella typhosa at 100°C for 30 minutes and Bs 20 (lmg of Salmonella typhosa per 1 ml of culture media) ?Salmonella typhosa 0.1 mg per ml. (100°C (60°C CYTOTOXIC ACTION TO THE CHICKEN FIBROBLAST IN VITRO 0.1 mg per 0.01 mg 0.01 mg Growth curve of 3 B) and 3 B’）Koktigen’： The culture media contain heat-kiled Salmonella typhosa O.Jmg per ml. (100 。Cfor 30 minutes) Vaccine : The culture media contain he"t-kiled Salmonella typhosa O.Jmg per ml. (60 °C for 30 minutes) This figure of Koktigen' does not mean true koktigen. Fig. 2 Growth curves of 3 A) and 3 A’）Koktigen' : The culture med ia containheat-kiled Salmonlla typhosa !mg per ml. (JOO℃ for 30 minutes) Vaccine : The culture media contain heat-kiled Salmonella typhosa I目l1g per ml.〔60°Cfor 30 minutes) This figure of koktigen' does not mean true koktigen. Fig. 1 ??，??〜?? 〜?????? 〜?．，? ???〜???ー 〜??．?、?、? ??、、，，?、。、????、??? 、，? ?、??? ??? ? 〜 ? ）? ? ? ?? 〜 ? ??? ? ???〈15 ．?，?? ?， ， ????， ， ?， ， ?， ， ?，?， ， ?， ， ?， ， ??， ， ??．?， ， ?????????????．?，???， ， ???? ? ? 〜??）??? ? ?? ??〜????????????5 3 Days of incubation Voce ine Kok fiocn’I Vaccine) Control Vacιl nc Koktiqcn’I Voce 1nc) Control 第4号4 A’） Two cc of Hanks 80 of the supernatant of the bactrial suspension contain-ing 1.25 mg of heat蝿killedSalmonella typhosa at 60°C for 30 minutes and Bs 20. 4 B) Two cc of Hanks 80 of the supernatant of the bacterial suspension contain-ing 0.125 mg of heat-killed Salmonella typhosa at 100°C for 30 minutes and Bs 20. 4 B’） Two cc of Hanks 80 of the supernatant of the bacterial suspension contain-ing 0.125 mg of heat-killed Salmonella typhosa at 60°C for 30 minutes and Bs 20. 4 C) Two cc of Hanks 80 of the supernatant of the bacterial suspension contain-ing 0.0125 mg of heat-killed Salmonella typhosa at 100°C for 30 minutes and Bs 20. 4’C) T vrn cc of Hanks 80 of the supernatant of the bacterial suspension contain-ing 0 0125 mg of heat-killed Salmonella Typhosa at 60°C for 30 minutes and Bs 20. 第31巻日本外科宝函638 RESULTS EXP ER I恥1ENTALGrowth curves of 4 A) and 4 A’） Koktigen : The culture media consist of the supernatant of the bacterial suspension containing 1皿gof heat-kiled Salmonella typhosa at l00°C for 30 minutes per ml. Vaccine' : The culture media consist of the supernatant of the bacterial suspension 1略。fhea-tkilled Samonella typhosa at 60℃ for 30 minutes per ml. Fig. 4 The五rstexperiment : Growth curve of 3 C and 3 C') Koktigen' : The culture media contain heat-kiled Salmonella typhosa 0心lmgper ml. (100。Cfor 30 minutes) Vaccine: The culture media contain heat-kiled Salmonella typhosa O.Olmg per ml. (60。Cfor 30 minutes) This figure of Koktigen does not mean true koktigen. Fig. 3 15 〔町、にミ、〉く） 、34ミミ吋』._ さえ10～ 、J、“ふミミミ～ 、、品、.＞、ド" '-、与-c:,. Eミミ3~ 〈， ， ?， ， ?， ， ?， ， ?， ， ?， ， ?， ， ?， ， ?， ， ??， ， ?， ， ?， ， ?， ， ???????????????????? ???，? ?? 》??????）?? ? ??? ?? ? 〜 ??? ?????」 ????? ? ? ? ? 〈5 3 Days of incubation V()aine' Kokti9cn Control 5 3 Days of incubolion Vaci1l Kol1!i9c目、（Vaccine〕Control In the series of bacteria-containing groups, both vaccines in each concentration reveal-el marked degenerations on the chicken fibroblasts, presenting vacuolization, cytolysis, deformation, and finally total break-down of the cells. In the comparison with the both vaccine, the toxic action to were no marked differences. In the experimental seri白 containingno bacteria, 2 A) and 2 A’） , 2 B) revealed slight toxic actions to the五broblasts,but 2 C) and 2 C’） revealed no differences in the phase contrast microscopy as compared with the control culture. The S配ondexperiment : In the series of bacteria containing groups, number, but rather decreased in number in the pr田ence639 the fibroblasts, there and 2 B’）marked CYTOTOXIC ACTION TO THE CHICKEN FIBROBLAST IN VITRO not mcrease m tthe concentrated did In the五broblasts of vaccines. Growth curves of 4 C and 4 C'J Koktigen・Theculutre media consist of the supernatant of the bacterial suspension containing 0.01 mg of heat kiled Salmonella typhosa at 100℃ for 30 minutes per ml. Vaccine' : The culture media consist of the supernatant of th bacterial suspension containing O.Olmg of heat kiled Salmonella typhosa at 60℃ for 30 minutes per ml. Fig. 6 Growth curve of 4 B and 4 B’）Koktigen : The culture media consist of the supernatant of the bacterial suspeusion conaining O.lmg of heat kiled Samonella typhosa at 100℃ for 30 minutes per ml. Vaccine’： The culture media consit of the supernatant of the bacterial suspensian containing O.lmg of heat”kiled Salmonella typhosa at 60℃ for 30 minutes per ml. Fig. 5 15 ?，??????．????? ?????????，??， ， ，??????? ????????，?，?? ??? ?15 ??????????? ? ? ? ? ??????? 、 ? ?5 3 Days of incubation Vaccine' Koktiqen Control 5 3 Days of incubation Vi ci ne’ Koktiqe1, 向ntrol640 日本外科宝函第31巻第4号culture media of 3 A), 3 A’） and 3 B), 3 B’）, the differences between the supernatant of the vaccine and the koktigen was not revealed, but in the dilute concentration of bacterial solutions, the toxic action koktigen were les than those of heated at 60° C for 30 minutes. In the series of the experiment containing no bacteria, 4 A) , 4 A’） and 4 B) and 4B’） revealed slight toxic action to the五broblasts,whereas in the groups of dilute con-centration of 4 C) and 4 C') no toxic actions to the fibroblasts were noted, but rather a growth promoting effect. DISCUSSION It is considered that Salmonella typhosa has an endotoxin, consisting of phospholipid, pol)明白haride,and protein. These components show different immunological behaviors and toxic action. The endotoxin acts as a pyrogen and the bacterial五ltrate国 U蜘Shwartzman phenomenon which contains cytotoxic action when introduced into animals, causing edema, redn問， necrosisin the local site of injection, and hemorrhage in the liver, kidney and bone marrow. The effect of heating on the Salmonella typhosa are assumed to伺 usethe partial inactivation of antigenicity of the bacteria, but the detailed bi配hemi-cal changes are not known. TORIKATA and his coworkers studied the heat killed bacterial vaccine and koktigen mainly by heating at 10° C for 30 minut回 haverev回 ledthe most active antibody producing and phag町yticaction to the body. In this experiment, it was proved that the supernatant of the heat killed bacterial suspensions had slight toxic action to the chicken fibroblasts but in the dilute concentration it had a favourable efefct to the growth of chiken fibroblast. The fietrates of the bacterial culture suspensions have many enzym田 andamino acid and polypeptides. From the biochemical point of view; this requires further investigations. CONCLUSIONS This paper deals with the toxic action to chicken fibroblast in vitro. The morpho-logical changes were observed by phase contrast microscope and the toxic action to the growth of the chicken fibroblasts were estimated by the Y oungners and citric crystal violet method. The results obtained are as follows : 1) The heat killed bacterial suspensions of Salmonella typhosa revealed the marked cytotoxic actions to the cultured chicken fibroblasts in the dilute concentration of 0.01 mg per ml of both vaccine and koktigen. 2) The supernatants of the heat killed bacterial suspensions also had slight toxic actions to the chicken fibroblasts in the concentration of 0.1 mg and 1 mg of Salmonella typhosa per ml, but inthe concentration of 0.01 mg of Salmonella typhosa per ml of the culture media, the supernatant fluid had a growth promoting action to the cels. In the comparison of vaccine and koktigen, the later exhibited les toxic actions and much more growth promoting actions to the chicken fibroblasts in vitro than the former. REFERENCES 1〕 DensenbyoKenkyujo Gakuyukai. : The Hand Book of Microbiological technique. Maruzen Co. CYTOTOXIC ACTION TO THE CHICKEN FIBROBLAST IN VITRO 641 Control groups Fig. 1 The chicken fibroblasts of the 3rd day after incubation. Fig. 3 The chicken fibroblasts of the 5th day after incubation. The experimental groups of 1 C’j Fig. 2 The chicken fibroblasts of the 5th day after incubation. Fig. 4 The chicken fibroblasts of the 5th day after incubation. The experimental culture media containing heat-killed Salmonella typhosa O.Olmg per ml (60°C tor 30 minutes) Fig. 5 Tissue culture of chicken fibroblasts before medium exchange. Fig. 6 Tisue culture of the Ist dιり afteradding the experimental medium. 642 日本外科宝画第31巻第4号Fig. 7 Tisue culture of the 3rd day after adding the experimental medium. The experimental groups of 1 C) Fig. 8 Tissue culture of the 5th day after adding the experimental medium. The experimental culture modia containing heat-killed Salmonella typhosa O.Olmg per ml. (100℃for 30 minutes) Fig. 9 Tissue culture of chicken fibroblasts before medium change. Fig. 11 Tissue culture of the 5th day after adding the experimetal medium. Fig. 10 Tissue culture of the Ist day after adding the experimental medium. Fig. 12 Tissue culture of the 3rd day after adding the experimental medium. CYTOTOXIC ACTION TO THE CHICKEN FIBROBLAST IN VITRO 643 Control groups Fig. 13 The tissue culture of the chicken fibroblasts before medium change. Fig. 15 Tissue culture of the 3rd day after medium change. The experimental groups of 2 C' l Fig. 14 Tissue culture of the 1st day after medium change. Fig. 16 Tissue culture of the 5th day after medium change. The experimental media of the supernatant of the bacterial suspensions containing heat-killed Salmonella typhosa I mg ) 60℃ for 30 minutes) Fig. 17 Tissue culture of the chicken fibroblasts before medium change. Fig. 18 Tissue culture of the !st day after adding the experimental media. 644 日本外科宝目白第31巻第4号Fig. 19 Tissue culture of the 3rd day after adding the experimental media. The experimental group of 2 AJ Fig. 20 Tissue culture of the 5th day after adding the experimental media. The experimental culture media of the supernatant of the bacterial suspensions containing heat-killed Salmonella typhosa 1皿g(100℃ for 30minutes) Fig. 21 Tissue culture of chicken fibroblasts before medium change. Fig. 23 Tissue cultuce of the 3rd day after adding the experimental media. Fig. 22 Tissue culture of the !st day after adding the experimental culture mediae. d、、正Fig. 24 Tissue culture of the 5th day adding the experimental media. CYTOTOXIC ACTION TO THE CHICKEN FIBROBLAST IN VITRO 645 Tokyo, 1957. 2) Higashi, H. : The Pathological Microbiology. Igakushoin,. Tokyo, 1959. 3) Katsuda, H. : Tissue Culture Technique. Naya Sheten .Tokyo, 1955. 4) Kimura, R : Tissue Culture as applied especially within Bacteriology and Immunology. Ejnar Munks-gard, Cophehagen, 1953. S) Ozu. S. : E"perimentelle Erforschung iiber die Gewinnung allgemeiner aktiver Immunitat mittels kutaner Applikation der Immunogene als Salben. Arch. Jap. Chir. 12, 1478, 1937. 6) Straus, N .Hendee, E. D. : J.E九p.Med .109, 145, 1959. 7) Torikata, R.: Koktopraezipitinogene u. Koktoimmunogen. Berlin 1917. 和文抄録培養鶏胎線維芽細胞に対するコクチゲン及びワクチンの毒性について京都大学医学部外科学教室第2講座（主任：青柳安誠教授）天 野 武 彦帰化後JO日の鶏胎児より試験管内スライド培養法に O.QJ凹1g/ccの濃度に於て著明な毒性を示した．よって猶殖した線維芽細胞に対するチフス菌加熱抗原 2) 100。C30分間及び60℃30分間加熱したチフス菌液の毒性乞位相差顕微鏡を用いて観察しp 叉Youn- の菌体を含まない上清成分を加えた培養に於ては，gner法によって線維芽細胞の増殖を定量的に追及し次 1昭／cc，及びO.lmg/ccの高濃度に於て，鶏胎線維芽細の結果を得た． 胞に対して軽度の増殖抑制作用を示したが，低濃度に1）菌体を含むチフス菌の 100℃30分間p 及び60℃ 於てはp むしろ細胞増殖促進作用を示した．特にコク30分間加熱ワクチンを含むHanks液80%(0.5% Lacto- チゲンは60℃30分間加熱の菌体を含まない上清成分とalbumin lydiolysateを含む），牛血清209五の培養液 比較してP 細胞に対する毒性は少かった．は，鶏胎線維芽細胞に対して， 1略／cc,O.lmg/cc及び

培養鶏胎線維芽細胞に対するコクチゲン及びワクチンの毒性について

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培養鶏胎線維芽細胞に対するコクチゲン及びワクチンの毒性について

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