Autoradiographic study on the bone marrow proliferation in experimental hypersplenism
비장의 기능항진이 골수에 미치는 영향을 추구하고자, egg-albumin 반복투여방법에 의
거 가토에게서 실험적 비기능항진증을 유발시켜 혈액학적 관찰을 하고, 골수를 채취하여
in vitro로 배양하였으며, 동시에 액성인자의 존재 가능성을 보기 위하여 정상 및 비기능
항진 혈청을 혼합 배양하고, H**3-thymidine(H**3-Th.)을 이용한 자기방사법을 시행하여
다음과 같은 결과를 얻었다.
Egg-albumin의 투여로 현저한 비종대와 함께 빈혈과 혈소판감소가 초래되었으나, 망상
구와 백혈구는 별 변화가 없었다. H**3 -Th.표지율은 비기능항진골수배양군에서 정상 및
비적출 골수배양군에 비하여 현저히 낮았고, 정상골수배양군에 비기능항진혈청을 혼합배
양하면 감소하였다. 반대로 비기능항진골수배양군에 정상혈청을 혼합 배양하면 증가하였
다. H**3-Th.표지치는 정상 및 비기능항진 골수배양군이 서로 유사하였으며, 비적출골수
배양군에서는 훨씬 낮았다. 그러나 정상과 비기능항진 혈청을 혼합배양한 군 사이에는 별
차가 없었다. 추산된 DNA 합성시간은 각 골수배양군간에 별 차이가 없었으나 비적출골수
배양군에서는 단축되었다. 세포생산시간은 비기능항진골수배양군에서 현저히 연장되었고,
비적출골수배양군에서는 약간 단축되었으며, 정상골수배양군에 비기능항진혈청을 첨가하
면 역시 현저하게 연장되었다. 그리고 정상 및 비기능항진 혈청을 혼합한 비기능항진골수
배양군에서도 현저하게 연장되었으나, 양군간의 차는 별로 없었다.
따라서 이상과 같은 결과를 종합 검토하면 비기능항진증에서의 혈구감소의 성인은 골수
세포의 증식과 성숙이 장애되기 때문이라 추리되며, 이러한 억제작용은 아마도 종대된 비
장에서 유리되어 혈액내에 존재하고 있는 어떤 액성인자에 기인하는 것이라 생각된다. 그
러나 이 기전만이 혈구감소의 유일한 성인이라고 단정할 수는 없다 하겠다.
[영문]The prob1em in hyperactivity of the spleen, since the day of Gretzel (1866) and
Banti (1882), has been plagued by uncertainty of meaning, doubt of etiology and
capriciousness of therapeutic results. In 1907, Chauffard introduced a term
"hypersplenism" and Dameshek in 1941 divided it into primary and secondary:
selective and total types.
The term encompasses various disorders in which blood cytopenia is associated
with splenomegaly, thus the cardinal features of the hypersplenism are: (1)
splenomegaly, which may be of unknown etiology (primary) , or secondary to some
known disease processes for splenic growth: (2) a reduction of one or more cellular
elements of the blood leading to anemia, leukopenia, thrombocytopenia, or any
combination of these : (3) hypercellular marrow with hyperplasia of the respective
marrow precursors of the deficient cell type : and (4) temporary or permanent
correction of the blood cytopenia (5) by splenectomy, even if it does not alternate
the basic disease process.
Since the Banti's hypotheses that the spleen destroys large number of blood
cells, or that certain biochemical processes taking place in the spleen results in
the development of certain metabolites which enter the circulation and have
secondary effects on hematopoietic organs, the controversy over the pathogenesis of
hypersplenism continues until the present time, and the hematology literature still
carried extensive debate and laboratory investigation supporting one or the other
of these two possibilities. Thus Dameshek (1941, 1955) concluded that the spleen
releases humours which suppress the production or release of new blood cells from
the marrow into circulation, whereas Dean (1949) argued that large spleens have an
enhanced capacity for sequestering blood cells, thereby lowering the peripheral
blood counts of these elements, and Rambach & Alt (1962) proposed an autoimmune
concept viewing hypersplenism as manifestations of autoimmune diseases.
Many attempts to induce hypersplenism in experimental animals have been
conducted, such as ligation of splenic and gastric coronary veins. administration
of silica gel, polyvinyl alcohol, gum acasia, pectin, hydroxylamine, antirabbit
marrow serum, tubercle bacilli, tryptophan blue, zymosan, methylcellulose, human
gamma-globulin and egg-albumin, but the autoradiographic technique in the study of
experimental hypersplenism has been utilized seldomly.
Therefore, the purpose of present study is to observe the effect of egg-albumin
induced hypersplenism on the bone marrow proliferation with autoradiographic
technique using H**3 -thy-midine in rabbits.
Materials and Methods
Hematologic observation: Rabbits weighing about 2kg, regardless of sex, were
divided into three groups : normal control, hyperaplenic and asplenic. The
hypersplenic group received daily injections of 1% egg-albumin solution 1ml and
asplenic group treated same way from two weeks after splenectomy. Hematologic
examinations including hemoglobin, hematocrit, reticulocyte, leukocyte and platelet
count were done with approximate;y two weeks interval.
Autoradiographic observations : On 97th day after injections of egg-albumin, the
bone marrow was obtained by femoral puncture under ether anesthesia, aspirated into
a heparinized syringe and immediately diluted with the same volume of culture
medium 199-Ⅸ (Earle's base). One ml of the bone marrow suspension was transferred
into pre-siliconized culture tubes. According to the following bone marrow culture
groups- (1) normal bone marrow (NM), (2) hypersplenic bone marrow(HSM), (3)
asplenic bone marrow(ASM) : (4) normal bone marrow with normal serum(NM+7NS) , (5)
normal bone marrow with hypersplenic strum(NM+HSS) : (6) hypersplenic bone marrow
with normal serum (HSM+NS), and (7) hypersplenic bone marrow with hypersplenic
serum (HSM+HSS) group, 0.5 ml of normal or hypersplenic serum obtained by cardiac
puncture at the time of bone marrow aspiration was added respectively, and then
H**3-thymidine (0.062 mg, 1 m1/1 mCi) was added to a final concentration of 2.5μCi
per milliliter. The procedure was carried out under sterile conditions and earth
bone marrow culture group was consisted of 3 culture tubes. Incubating the tuhea at
37℃., samples taken from each tube at 1, 3, 5, 10, and 24 hours smearer on
gelatin-coated slides, fixed in Carney's solution, and after several washing in
distilled water, autoradiographic preparations were made with Kodak Fine-Grain
Autoradiographic Stripping Plate AR-10 according to the technique described by Pelc
(1956). After an exposure of 7 days, the firms were developed and stained through
stripping film with Giemsa.
One thousand nucleated cells observed and grains over the cell nucleus was
estimated as scores as table 1 in the text. Fer each autoradiograph, (1)
differential count of cells, (2) per cent labeled cells, and (3) scores per labeled
cell were counted, then (4) DNA synthetic time and (5) generation time for each
cell type in each group were deduced.
Results and Conclusions
The results obtained are summarized as follows:
1. Repeated intravenous injecitons, of egg-albumin to rabbits induced
splenomegaly, anemia and thrombocytopenia without significant changes of
reticulocyte and leukocyte count, whereas only mild thrombocytopenia was noted in
2. There was no significant difference of differential cell count in each group.
3. Per cent labeled cells was markedly reduced in HSM group and somewhat
increased in ASM group. It was also reduced in NM+HSS group while there was no
change in NM+NS group. Though it was decreased in HSM+NS group and HSM+HSS group,
it twas higher in the former.
4. Scores per labeled cell baa similar in NM and HSM group, but significantly low
in ASM group. There was no significant difference either between NM+NS and NM+HSS
group, or HSM+NS and HSM+HSS group.
5. DNA synthetic time showed no significant difference between the groups, but it
was shortened in ASM group.
6. Generation time was significantly prolonged in HSM group and slightly
shortened in ASM group. It was also markedly prolonged in NM+HSS group while there
was no change in NM+NS group. There was marked prolongation of it in both HSM+NS
and HSM+HSS group, but no significance between these two groups.
7. Such differences observed with autoradiographic technique were remarkable in
the erythroid elements but indistinct in the myeloid elements.
The results obtained from present study indicate that the cytopenias induced by
egg-albumin administration in rabbits are mostly due to defects in proliferation
and maturation of the bone marrow cells-especially erythroid series, and this
inhibitory effect nay be attributed to possible humoral factor(s) released from the
enlarged spleen and present in the serum. But it cannot be concluded whether this
inhibitory effect is the only mechanism far the cytopenia(s) in hypersplenism or